Next Article in Journal
The Spatiotemporal Evolutionary Trend and Driving Factors of the Coupling Coordinated Development between Regional Green Finance and Ecological Environment
Next Article in Special Issue
Level of Serum Fetuin-A Correlates with Heart Rate in Obstructive Sleep Apnea Patients without Metabolic and Cardiovascular Comorbidities
Previous Article in Journal
Text Mining Analysis of Korean University Students’ Academic Coaching Intake Session Reports
Previous Article in Special Issue
A Retrospective Cohort Study of the Association between Serum Osteopontin Levels and Aortic Stiffness in Hypertensive Patients
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJERPH
Volume 19
Issue 10
10.3390/ijerph19106210
ijerph-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Effect of Reconstructive Procedures of the Extracranial Segment of the Carotid Arteries on Damage to the Blood–Brain Barrier

by
Piotr Terlecki
1,
Stanisław Przywara
1,
Karol Terlecki
1,*,
Dariusz Janczak
2,
Maciej Antkiewicz
2 and
Tomasz Zubilewicz
1
1
Department of Vascular Surgery and Angiology, Medical University of Lublin, 20-078 Lublin, Poland
2
Department of Vascular Surgery, General and Transplant Surgery, Medical University in Wroclaw, 50-355 Wroclaw, Poland
*
Author to whom correspondence should be addressed.
Int. J. Environ. Res. Public Health 2022, 19(10), 6210; https://doi.org/10.3390/ijerph19106210
Submission received: 21 March 2022 / Revised: 16 May 2022 / Accepted: 18 May 2022 / Published: 20 May 2022
(This article belongs to the Special Issue Vascular Disease and Health)
Browse Figures
Versions Notes

Abstract

:
Introduction: Endarterectomy and angioplasty of the internal carotid artery are surgical measures for the prevention of ischemic stroke. Perioperative complications are caused by concomitant embolism and reperfusion syndrome leading to damage of the blood–brain barrier. Methods: The study included 88 patients divided into two groups, depending on the surgical technique used: internal carotid artery endarterectomy (CEA), 66 patients, and percutaneous carotid angioplasty and stenting (CAS), 22 patients. Blood was drawn 24 h before surgery, as well as 8, 24, and 48 h post-surgery. The assessment of damage to the blood–brain barrier was based on the evaluation of the concentration of claudin-1 and occludin, aquaporin-4, the measurements of the activity of metalloproteinase-2 (MMP-2) and -9 (MMP-9), and the assessment of central nervous system damage, measured by changes in the blood S100β protein concentration. Results: A significant increase in the concentration of the blood–brain barrier damage markers and increased MMP-2 and MMP-9 activity were found in patient blood. The degree of damage to the blood–brain barrier was higher in the CEA group. Conclusions: The authors’ own research has indicated that revascularization of the internal carotid artery may lead to damage to the central nervous system secondary to damage to the blood–brain barrier.

1. Introduction

Stroke is the most common cause of severe disability in adults and the third leading cause of death, after heart disease and cancer, in highly industrialized countries [1,2]. Approximately 20–30% of cerebral ischemic strokes are caused by advanced atherosclerotic lesions that cause internal carotid stenosis, but only 5% require interventional treatment. The degree of stenosis in the artery and the function of collateral circulation through the circle of Willis are critical [3,4].
Damage to the brain during ischemic stroke is mainly caused by hypoxia of the nerve cells [5]. Inflammatory reactions in parenchyma have also been reported, initiated by its hypoxia. Other pathological lesions include damage to the blood–brain barrier (BBB). The main factors damaging the BBB are intercellular matrix metalloproteinases released by inflammatory cells, among others, cells damaging the basal membrane, pro-inflammatory factors and free radicals secreted by inflammatory cells, as well as by mechanical factors and hypoxia damaging the vascular endothelium. Cerebrovascular edema occurs, which causes swelling of the nerve tissue and increased intracranial pressure [6,7].
The research data suggest that both intraoperative microembolism and reperfusion syndrome are conducive to central nervous system injury through BBB damage. These factors may cause cerebral edema due to BBB damage and increased fluid permeability through the barrier [8,9,10,11].
Animal studies have shown that the increased transmissibility of the BBB has been caused by abnormally higher activity and expression of matrix metalloproteinases, such as metalloproteinases 2 (MMP-2) and metalloproteinases 9 (MMP-9). Significant increases in MMP-9 activity were also found in individuals who had a previous stroke [12,13,14]. Panickar’s studies [15] have shown that MMP-9 modified the permeability of BBB by the degradation of its components, such as tight junction proteins including occludin and claudin-5. Occludin is critical to the barrier function at the site of the tight junctions between microvessel endothelial cells. Moving outside of the tight junctions is a sensitive, early, and reliable signal of the opening of the tight junctions and BBB dysfunction [16]. Aquaporin-4 plays an important role in the pathogenesis of cerebral edema, as its molecule is the most efficient aqueous channel in this area, regulating its transmembrane flow in astrocytes [17].
S100β protein is one of the best-known biomarkers of central nervous system damage. The low molecular weight of S100β protein causes it to permeate the blood–brain barrier even with a minor pathology, and even a relatively small activation of astrocytes increases its synthesis and penetration into the periastrocytic space. Elevated blood concentrations observed in stroke patients may indicate an ongoing release of the marker by necrotic tissue [18].
The objective of this study was to evaluate the activity of MMP-2 and MMP-9 as well as the concentrations of claudin-1, occludin, aquaporin-4, and S100β protein in the blood of patients undergoing extracranial carotid reconstructive surgery.

2. Materials and Methods

The study was conducted based on the approval of the Bioethics Committee (KE-0254/146/2010).
A total of 88 elective patients were enrolled in the study due to extracranial stenosis of the internal carotid arteries, which qualified for interventional treatment according to the guidelines of the European Society for Vascular Surgery [19] based on Doppler testing performed with a Toshiba Aplio 500 instrument. The morphological nature (stability) of the atherosclerotic plaque and the degree of stenosis were evaluated based on the North American Symptomatic Carotid Endarterectomy Trial (NASCET) study recommendations [20]. The mean age of the patients was 67 (53–83) years old. The mean stenosis of the right internal carotid artery was 75% (50–90%), and the left, 80% (50–95%). Patients were divided into two groups depending on the surgical technique used: internal carotid endarterectomy (CEA), 66 patients, and endovascular percutaneous internal carotid balloon angioplasty and stenting (CAS), 22 patients. The mean time of surgery was 67 ± 14 min in the CEA group and 61 ± 10 min in the CAS group, respectively. The cross-clamping time was 11 (5–39) min. No local or systemic surgical complications were observed.
Blood for laboratory assays was drawn from the basilic vein 24 h prior to surgery (measurement 1), 8 h post-surgery (measurement 2), 24 h post-surgery (measurement 3), and 48 h post-surgery (measurement 4). After centrifugation at 3500 rpm for 5 min, the samples were frozen and stored at −80 °C. Samples for assays were thawed in a water bath at 37 °C.
Claudin-1, occludin, aquaporin-4, and S100B concentrations were assayed with pre-filled reagent sets using the ELISA assay, and MMP-2 and MMP-9 activity was evaluated using zymography. Determination of gelatinases was made using the G BOX gel analysis and documentation system by comparing the locations of the bands obtained both with a protein pattern of known molecular weight and with a location relative to the control MMP-2 and MMP-9. Quantitative analysis was performed using Gene Tools computer software (Figure 1).
The results of the studies were analyzed statistically. Due to the skewed distributions of the tested measurable parameters as assessed by the Shapiro–Wilk test, and the heterogeneity of variance assessed by Levene’s test, to analyze the differences between the studied subgroups nonparametric tests were used. The two independent groups were compared with the Mann–Whitney U test, while more than two independent groups were compared with the Kruskal–Wallis H test and for the dependent groups, Friedman’s ANOVA test and post hoc multiple comparisons were used. The significance level was set at p < 0.05. Statistical analyses were performed based on computer software STATISTICA v. 10.0 (StatSoft Inc., Tulsa, OK, USA).
Patient parameter characteristics are presented in Table 1.

3. Results

A significant decrease in MMP-2 activity in the blood was found in all subjects undergoing endarterectomy. Most significant changes were recorded immediately after the surgery and in the morning on the first postoperative day (p < 0.01) (Figure 2).
A significant increase in MMP-9 activity in the blood was found in the endarterectomy group and applied to all studied patients. As with the MMP-2 analysis, the largest changes were observed immediately post-surgery (p < 0.001) and on the first postoperative day (p < 0.01) (Figure 3).
Analysis of MMP activity 2 and 9 showed no significant differences between patients in the CEA and CAS groups immediately prior to surgery (p > 0.05). Significantly higher MMP-2 activity was noted in patients undergoing angioplasty, but only immediately after the surgery (p < 0.05) (Figure 4).
An analysis of the changes in claudin-1 concentrations in all studied patients showed a significant increase on the first and second postoperative day (Figure 5). Significantly higher claudin-1 concentrations were reported in patients who underwent percutaneous carotid artery stenting (CAS) (p < 0.05).
A significant increase in occludin concentrations was observed in all subjects only during the near-postoperative period (p < 0.01) (Figure 6). On the first and second postoperative days, regardless of the type of procedure performed, the concentration of the tested parameter was close to the baseline values. Significantly higher occludin concentrations were found on the first postoperative day (p < 0.05) in the group of patients who underwent carotid endarterectomy (Figure 7).
Cerebral circulatory revascularization resulted in a significant increase in blood plasma aquaporin-4 concentration in both groups. Most significant increases/changes were recorded on the second postoperative day (p < 0.001) (Figure 8). An analysis of the aquaporin-4 concentration based on the type of surgery did not show statistically significant differences between the studied groups (p > 0.05).
Both studied groups showed a significant reduction in the concentration of S100β protein within a short time after the procedure (p < 0.01). Within the first postoperative day, the concentration of S100β protein increased significantly and then decreased on the second postoperative day (p < 0.01) (Figure 9, Table 2). Significantly higher S100β protein concentrations were reported in the group of patients undergoing endarterectomy before and immediately post-surgery (p < 0.05).
No statistically significant differences were found with respect to patient demographic data (p > 0.05) when analyzing the changes in the tested parameters depending on the stability of the atherosclerotic plaque. Significantly higher MMP-9 activity was found prior to surgery in the group of patients with unstable atherosclerotic plaque (p = 0.05), and the MMP-2 activity was comparable across all groups and throughout the observation period. In the group of patients with unstable atherosclerotic plaque, significantly higher S100β protein concentrations were found prior to surgery (p < 0.05). The concentrations of the remaining tested parameters were similar in the group of patients with stable and unstable atherosclerotic plaque (Table 3).

4. Discussion

Open carotid endarterectomy (CEA) is an effective treatment for internal carotid artery stenosis in its extracranial segment. It is the basis for stroke prevention in patients with symptoms of cerebral ischemia as well as in asymptomatic patients. The alternative for this treatment is percutaneous angioplasty and stenting; however, numerous clinical studies conducted to date have indicated that the surgical method has been associated with fewer serious perioperative neurological complications [21,22,23,24]. Wu et al. reported that early revascularization, including revascularization within seven days, may be performed safely. It was considered to be a better course of management than the delayed surgical treatment of symptomatic carotid stenosis in patients without signs of hemorrhagic stroke, carotid artery occlusion, or deep neurological deficit [25]. This has also been confirmed by the authors’ experience in 20 endarterectomies and thrombectomies or embolectomies performed during the “stroke in evolution” period [26]. In Rapp’s study [27], which analyzed 54 internal carotid angioplasty procedures with excellent clinical results, a significant number of new lesions in diffusion weighted magnetic resonance imaging (DW-MRI) were found. These subclinical brain injuries did not occur during the procedure but in the subsequent 48 h when transcranial Doppler examinations confirmed an active embolic process. According to Feiwell, new lesions were rarely observed in MRIs after endarterectomy [28]. Comparing the CAS and CEA procedures, Roh and Szarmach [29,30] demonstrated that both neurological events as well as new lesions in the MRI radiographic image were more common in patients undergoing endovascular procedures.
Ischemia and hypoxia of the central nervous system trigger a number of events leading to the interruption of occluding junctions and increased BBB permeability. They appear to be mediated by cytokines, vascular endothelial growth factor (VEGF), and nitric oxide (NO). Following focal and generalized cerebral ischemia in animals and in cerebrospinal fluid, post-stroke patients demonstrated elevated concentrations of proinflammatory cytokines, IL-1 and tumor necrosis factor (TNF). In the experimental BBB in vitro model comprising human endothelial cells and astrocytes, it was found that induced ischemia stimulated the secretion of IL-8 and MCP-1 from endothelial cells and astrocytes [31,32,33]. Oxidative stress damaged endothelial cells forming the BBB and contributed to vasogenic edema. The superoxide radical (O2−) was identified as the primary reactive form of oxygen contributing to increased blood vessel permeability and the formation of edema in generalized and focal cerebral ischemia [34]. BBB damage is a progressive process. Initial ischemia injury progresses through ischemia via a reperfusion mechanism [35]. BBB damage associated with reperfusion has been the subject of extensive research, which has led to the identification of multiple mechanisms that contribute to the reperfusion damage of the BBB, such as oxidative trauma, inflammatory damage, vascular activation, or dysregulation of extracellular proteolysis. Unlike the reperfusion phenomenon, the initial stage of damage to the BBB resulting from ischemia has been a less studied topic [36]. Many studies using the BBB in vitro model have demonstrated that hypoxia and hypoxia/reoxygenation leads to increased permeability and/or a rupture of BBB-occluding junctions [37,38,39], although the stress associated with hypoxia may also increase transcellular permeability [40]. In vivo hypoxia has been associated with increased BBB permeability, reduced occludin expression, and the activation of nuclear transcription factors NF-kB and hypoxia-induced factor [41].
Numerous studies have suggested that MMP-9 played a key role in the modulation of BBB permeability in various conditions such as head injury, acute liver failure, focal or generalized ischemia, and reperfusion in the central nervous system. Yang’s studies have shown that the development of cerebral edema and increased BBB permeability in the early period following an endarterectomy of the internal carotid artery and the restoration of normal circulation resulted in increased MMP-9 activity and expression, and a reduction in the number of proteins in tight junctions, including claudin-5 and occludin. It was established that the disturbance of the structure of the tight junctions in the barriers was directly related to the pathogenesis and worsening of cerebral ischemia and post-traumatic reperfusion [42]. MMP-9 has been linked to BBB permeability by modulating the proteins of the tight junctions and basal plaque combinations, which confirmed that the increased MMP-9 expression was accompanied by a reduction in basal lamina proteins including laminin and fibronectin. That literature has also suggested that increases in MMP-9 activity and its expression correlated with a decrease in claudin-5 and occludin levels, suggesting that the regulation of BBB permeability by MMP-9 may occur by the degradation of the tight junction proteins. Given that the matrix metalloproteinases are a family of enzymes that are activated by oxidative stress, and ischemic post-conditioning may inhibit oxidative stress caused by the release of the carotid stenosis, we speculated that the inhibitory effect of ischemic post-conditioning on MMP-9 could be caused by blocking oxidative stress, which was also confirmed by studies where the administration of a free radical scavenger inhibited BBB damage [43,44]. MMP-2 is the primary gelatinase secreted by ischemic cerebral tissue two hours after middle cerebral artery occlusion (MCAO), which was consistent with the assumption that MMP-2 was an early mediator in ischemic brain injury, and MMP-9 was an inflammatory molecule stimulated and involved in brain damage at relatively late stages of stroke [45]. The MCAO in vivo model also showed that cerebral ischemia rapidly induced the degradation of occludin and redistribution of claudin-5 in ischemic cerebral microvasculature [46].
The authors’ research has shown a significant increase in MMP2 and MMP9 activity and a significant increase in the concentrations of claudin-1 and occludin in the blood of patients undergoing the reconstruction of internal carotid artery stenosis. Due to the involvement of the above parameters in the damage to the blood–brain barrier and cerebral ischemia, the results obtained suggested the occurrence of the above processes in the studied patients.
S100B protein has been considered a sensitive marker of cerebral trauma [47,48]. In the periodical Doppler (TCD) examination, during carotid revascularization procedures, a significant number of high intensity transient signals (HITS) consistent with embolisms correlated with S100β concentration changes were found. The CAS procedure has been associated with a higher number of perioperative HITS, as compared to the CEA procedure (despite the routine use of neuroprotection systems); however, no statistically significant changes were observed in S-100b concentrations when comparing both methods. A statistically insignificant, but apparent, tendency was observed for the mean concentration of S100β to increase in patients undergoing the CAS procedure, while the mean concentration of this marker in patients undergoing CEA remained constant throughout the duration of the procedure [49]. Capoccia et al. reported a postoperative increase in the concentration of S100β protein and neuron-specific enolase (NSE) in patients post-CAS and in all those demonstrating new lesions in the postoperative DW-MRI examination. These changes correlated with significant decreases in postoperative MMSE neuropsychometric tests with a tendency for some improvement at the six-month follow-up. In addition, a significant relationship was noted between the emergence of new ischemic lesions in the postoperative DW-MRI examination and the deterioration of MMSE results in patients post-CAS, as compared to preoperative values and the results of patients who did not demonstrate postoperative ischemic lesions [50].
Our research showed a statistically significant increase in S100β protein concentrations in the patients’ blood within the first postoperative day. Significantly higher concentrations of this parameter were observed in patients treated with endarterectomy prior to and immediately post-surgery, which may indicate cerebral ischemia following surgery.
Cerebral edema is considered an important factor of high morbidity and mortality due to extensive ischemic strokes. Solenov et al. demonstrated that astrocytes exhibiting aquaporin-4 deficiency were seven times less permeable to water as compared to identical cells with normal aquaporin-4 expression [51]. The exclusion of aquaporin-4 and the interference of the expression of its polarized parts reduced the accumulation of fluid in the brain associated with ischemia, water intoxication, and hyponatremia, suggesting that under pathophysiological conditions, aquaporin-4 was the main route of penetration of plasma water into the brain. Aquaporin-4 deficiency reduced cytotoxic edema caused by water intoxication, cerebral ischemia, and acute bacterial meningitis; however, it intensified vascular edema caused by brain tumor, the damage to the cortex due to hypothermia, central nervous system infections, and kaolin-induced obstructive hydrocephalus [52,53]. The data on the expression of aquaporin-4 in cerebral edema, particularly aquaporin-1 and aquaporin-4 that are expressed in the central nervous system and participate in the transport of water by the BBB, have been taken from several recent studies on extensive ischemic strokes [54]. The presence of aquaporin-4 molecules in the BBB suggested that the aquaporin-4 molecules may be important for maintaining water balance in the brain and may play a key role in the pathogenesis of its edema. Aquaporin-4 is found in the astrocytic processes between the brain tissue and larger fluid compartments, including spaces containing cerebrospinal fluid and blood. The aquaporin-4 degradation indicated that it played an important role in controlling the flow of water into and out of the brain tissue [55]. The importance of aquaporin-4 in the development of cerebral edema was confirmed by studies on the sepsis-associated encephalopathy model, which showed that a higher level of aquaporin-4 was directly associated with a more severe degree of edema [56]. Data from studies of aquaporin-4 conducted in knock-out mice suggested that an aquaporin-4 molecule was involved in the removal of extracellular fluid from brain tissues during vasogenic edema. The aquaporin-4 observations in the knock-out mice with water intoxication and focal cerebral ischemia suggested that during vasogenic edema, water entered the brain tissues regardless of aquaporin-4, but left the brain with aquaporin-4 [57]. The increase in aquaporin-4 concentration observed in the authors’ own research within the second postoperative day suggested significant disturbances in BBB permeability resulting in cerebral edema.

5. Conclusions

To summarize, the increase in the concentration of the BBB damage markers and central nervous system damage indicated that carotid endarterectomy may have led to injury in the central nervous system, secondary to damage to the BBB. Both techniques may have led to injury, but the degree of damage to the BBB was dependent on the surgical technique performed, and it was higher in the group of patients treated surgically with CEA.

Author Contributions

Conceptualization: P.T.; methodology: P.T.; software: M.A.; validation: P.T., K.T. and S.P.; formal analysis: P.T. and K.T.; investigation: P.T. and T.Z.; resources: D.J.; data curation: P.T. and K.T.; writing—original draft preparation: K.T. and S.P.; writing—review and editing: P.T. and K.T.; visualization: K.T.; Supervision: P.T.; project administration: P.T.; funding acquisition: P.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The study was conducted based on the approval of the Bioethics Committee (KE-0254/146/2010).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

Not applicable.

Acknowledgments

The authors would like to thank Agata Smoleń for developing the statistical analysis of the publication.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Sobieszczyk, P.; Beckman, J. Carotid artery disease. Circulation 2006, 114, e244–e247. [Google Scholar] [CrossRef] [PubMed]
  2. Andziak, P. Zwężenia tętnic szyjnych wewnętrznych. Chirurgia tętnic i żył pod redakcją W. Noszczyk PZWL 2007, 35, 485–507. [Google Scholar]
  3. Ederle, J.; Brown, M. The evidence for medicine versus burgery for carotid stenosis. Eur. J. Radiol. 2006, 60, 3–7. [Google Scholar] [CrossRef] [PubMed]
  4. Flint, A.C.; Bhandari, S.G.; Cullen, S.P.; Reddy, A.V.; Hsu, D.P.; Rao, V.A.; Patel, M.; Pombra, J.; Edwards, N.J.; Chan, S.L. Detection of anterior circulation large artery occlusion in ischemic stroke using noninvasive cerebral oximetry. Stroke 2018, 49, 458–460. [Google Scholar] [CrossRef]
  5. Lee, J.M.; Zipfel, G.J.; Choi, D.W. The changing landscape of ischaemic brain injury mechanisms. Nature 1999, 399, A7–A14. [Google Scholar] [CrossRef]
  6. Emerich, D.F.; Dean, R.L.; Bartus, R.T. The role of leukocytes following cerebral ischemia: Pathogenic variable or bystander reaction to emerging infarct? Exp. Neurol. 2002, 173, 168–181. [Google Scholar] [CrossRef]
  7. McIlvoy, L.H. The effect of hypothermia and hyperthermia on acute brain injury. AACN Adv. Crit. Care 2005, 16, 488–500. [Google Scholar] [CrossRef]
  8. Kim, K.A.; Kim, D.; Kim, J.H.; Shin, Y.J.; Kim, E.S.; Akram, M.; Kim, E.H.; Majid, A.; Baek, S.H.; Bae, O.N. Autophagy-mediated occludin degradation contributes to blood-brain barrier disruption during ischemia in bEnd.3 brain endothelial cells and rat ischemic stroke models. Fluids Barriers CNS 2020, 17, 21. [Google Scholar] [CrossRef] [Green Version]
  9. Klionsky, D.J.; Abdelmohsen, K.; Abe, A.; Abedin, M.J.; Abeliovich, H.; Arozena, A.A.; Adachi, H.; Adams, C.M.; Adams, P.D.; Adeli, K.; et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 2016, 12, 1–222. [Google Scholar]
  10. Kago, T.; Takagi, N.; Date, I.; Takenaga, Y.; Takagi, K.; Takeo, S. Cerebral ischemia enhances tyrosine phosphorylation of occludin in brain capillaries. Biochem. Biophys Res. Commun. 2006, 339, 1197–1203. [Google Scholar] [CrossRef]
  11. Lakhan, S.E.; Kirchgessner, A.; Tepper, D.; Leonard, A. Matrix metalloprotein–ases and blood–brain barrier disruption in acute ischemic stroke. Front. Neurol. 2013, 4, 32. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  12. Lok, J.; Zhao, S.; Leung, W.; Seo, J.H.; Navaratna, D.; Wang, X.; Whalen, M.J.; Lo, E.H. Neuregulin-1 effects on endothelial and blood-brain-barrier permeability after experimental injury. Transl. Stroke Res. 2012, 3, S119–S124. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Chen, F.; Ohashi, N.; Li, W.; Eckman, C.; Nguyen, J.H. Disruptions of occluding and claudin-5 in brain endothelial cells in vitro and in brains of mice with acute liver failure. Hepatology 2009, 50, 1914–1923. [Google Scholar] [CrossRef] [Green Version]
  14. Switzer, J.A.; Hess, D.C.; Ergul, A.; Waller, J.L.; Machado, L.S.; Portik-Dobos, V.; Pettigrew, L.C.; Clark, W.M.; Fagan, S.C. MatrixMetalloproteinase-9 in an Exploratory Trial of Intravenous Minocycline for Acute Ischemic Stroke. Stroke 2011, 42, 2633–2635. [Google Scholar] [CrossRef] [Green Version]
  15. Panickar, K.S.; Anderson, R.A. Effect of polyphenols on oxidative stress and mitochondrial dysfunction in neuronal death and brain edema in cerebral ischemia. Int. J. Mol. Sci. 2011, 12, 8181–8207. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Dobrogowska, D.H.; Vorbrodt, A.W. Immunogold localization of tight junctional proteins in normal and osmotically-affected rat blood-brain barrier. J. Mol. Histol. 2004, 35, 529–539. [Google Scholar] [CrossRef]
  17. Simard, M.; Arcuino, G.; Takano, T.; Liu, Q.S.; Nedergaard, M. Signalling at the gliovascular interface. J. Neurosci. 2003, 23, 9254–9262. [Google Scholar] [CrossRef]
  18. Kilminster, S.; Treasure, T.; McMillan, T.; Holt, D.W. Neuropsychological change and S-100 protein release in 130 unselected patients undergoing cardiac surgery. Stroke 1999, 30, 1869–1874. [Google Scholar] [CrossRef] [Green Version]
  19. Liapis, C.; Bell, S.P.; Mikhailidis, D.; Sivenius, J.; Nicolaides, A.; e Fernandes, J.F.; Biasi, G.; Norgren, L. ESVS guidelines. Invasive treatment for carotid stenosis: Indications, techniques. Eur. J. Vasc. Endovasc. Surg. 2009, 37, 1–19. [Google Scholar] [CrossRef] [Green Version]
  20. Staikov, I.N.; Arnold, M.; Mattle, H.P.; Remonda, L.; Sturzenegger, M.; Baumgartner, R.W.; Schroth, G. Comparison of the ECST, CC, and NASCET grading methods and ultrasound for assessing carotid stenosis. European Carotid Surgery Trial. North American Symptomatic Carotid Endarterectomy Trial. J. Neurol. 2000, 247, 681–686. [Google Scholar] [CrossRef]
  21. Goyal, M.; Menon, B.K.; Van Zwam, W.H.; Dippel, D.W.J.; Mitchell, P.J.; Demchuk, A.M.; Dávalos, A.; Majoie, C.B.L.M.; Van Der Lugt, A.; De Miquel, M.A.; et al. Endovascular thrombectomy after large-vessel ischaemic stroke: A meta-analysis of individual patient data from five randomised trials. Lancet 2016, 387, 1723–1731. [Google Scholar] [CrossRef]
  22. Baek, J.H.; Kim, B.M.; Kim, D.J.; Heo, J.H.; Nam, H.S.; Yoo, J. Stenting as a Rescue Treatment after Failure of Mechanica Thrombectomy for Anterior Circulation Large Artery Occlusion. Stroke 2016, 47, 2360–2363. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Kimmel, E.R.; Al Kasab, S.; Harvey, J.B.; Bathla, G.; Ortega-Gutierrez, S.; Toth, G.; Jaksich, E.M.; Sheharyar, A.; Roa, J.; Hasan, D.M.; et al. Absence of Collaterals is Associated with Larger Infarct Volume and Worse Outcome in Patients with Large Vessel Occlusion and Mild Symptoms. J. Stroke Cerebrovasc. Dis. 2019, 28, 1987–1992. [Google Scholar] [CrossRef] [PubMed]
  24. Constantinou, J.; Jayia, P.; Hamilton, G. Best evidence for medical therapy for carotid artery stenosis. J. Vasc. Surg. 2013, 58, 1129–1139. [Google Scholar] [CrossRef] [Green Version]
  25. Wu, T.Y.; Anderson, N.E.; Barber, P.A. Neurological complications of carotid revascularization. J. Neurol. Neurosurg. Psychiatry 2012, 83, 543–550. [Google Scholar] [CrossRef]
  26. Terlecki, P.; Wroński, J.; Przywara, S.; Zubilewicz, T.; Feldo, M.; Kobusiwicz, W.; Kęsik, J.; Iłżecki, M.; Kropski, W. Urgent and early carotid revascularization in selected patients with acute ischemic stroke. II Międzynarodowe Sympozjum Naukowe “Środowiskowe źródła zagrożeń zdrowotnych. In Kazimierz Dolny 2007; Wydawnictwo Polihymnia: Lublin, Poland, 2007; Volume 2, p. 665. [Google Scholar]
  27. Rapp, J.H.; Wakil, L.; Sawhney, R.; Pan, X.M.; Yenari, M.A.; Glastonbury, C.; Coogan, S.; Wintermark, M. Subclinical embolization after carotid artery stenting: New lesions on diffusion-weighted magnetic resonance imaging occur postprocedure. J. Vasc. Surg. 2007, 45, 867–874. [Google Scholar] [CrossRef] [Green Version]
  28. Feiwell, R.J.; Besmertis, L.; Sardar, R.; Saloner, D.A.; Rapp, J.H. Detection of clinically silent infarcts after carotid endarterectomy by use of diffusionweighted imaging. Am. J. Neuroradiol. 2001, 22, 646–649. [Google Scholar]
  29. Roh, H.G.; Byun, H.S.; Ryoo, J.W.; Na, D.G.; Moon, W.-J.; Lee, B.B.; Kim, D.-I. Prospective analysis of cerebral infarction after carotid endarterectomy and carotid artery stent placement by using diffusion-weighted imaging. Am. J. Neuroradiol. 2005, 26, 376–384. [Google Scholar]
  30. Arkadiusz Szarmach, G.H. Carotid Artery Stenting and Blood–Brain Barrier Permeability in Subjects with Chronic Carotid Artery Stenosis. Int. J. Mol. Sci. 2017, 18, 1008. [Google Scholar] [CrossRef] [Green Version]
  31. Feuerstein, G.Z.; Liu, T.; Barone, F.C. Cytokines, inflammation, and brain injury: Role of tumor necrosis factor-alpha. Cerebrovasc. Brain Metab. Rev. 1994, 6, 341–360. [Google Scholar]
  32. Tarkowski, E.; Rosengren, L.; Blomstrand, C.; Wikkelso, C.; Jensen, C.; Ekholm, S.; Tarkowski, A. Intrathecal release of pro- and anti-inflammatory cytokines during stroke. Clin. Exp. Immunol. 1997, 110, 492–499. [Google Scholar] [CrossRef] [PubMed]
  33. Zhang, W.; Smith, C.; Shapiro, A.; Monette, R.; Hutchison, J.; Stanimirovic, D. Increased expression of bioactive chemokines inhuman cerebromicrovascular endothelial cells and astrocytes subjected to simulated ischemia in vitro. J. Neuroimmunol. 1999, 101, 148–160. [Google Scholar] [CrossRef]
  34. Kim, G.W.; Gasche, Y.; Grzeschik, S.; Copin, J.C.; Maier, C.M.; Chan, P.H. Neurodegeneration in striatuminduced by the mitochondrial toxin 3-nitropropionic acid: Role of matrix metalloproteinase-9 in early blood-brain barrier disruption? J. Neurosci. 2003, 23, 8733–8742. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Iłżecki, M.; Iłżecka, J.; Przywara, S.; Terlecki, P.; Grabarska, A.; Stepulak, A.; Zubilewicz, T. Serum Neuron-Specific Enolase as a Marker of Brain Ischemia-Reperfusion Injury in Patients Undergoing Carotid Endarterectomy. Acta Clin. Croat. 2016, 55, 579–584. [Google Scholar] [CrossRef] [Green Version]
  36. Jung, J.E.; Kim, G.S.; Chen, H.; Maier, C.M.; Narasimhan, P.; Song, Y.S.; Niizuma, K.; Katsu, M.; Okami, N.; Yoshioka, H.; et al. Reperfusion and neurovascular dysfunction in stroke: From basic mechanisms to potential strategies for neuroprotection. Mol. Neurobiol. 2010, 41, 172–179. [Google Scholar] [CrossRef] [Green Version]
  37. Abbruscato, T.J.; Davis, T.P. Combination of hypoxia/aglycemia compromises in vitro blood-brain barrier integrity. J. Pharmacol. Exp. Ther. 1999, 289, 668–675. [Google Scholar]
  38. Fischer, S.; Clauss, M.; Wiesnet, M.; Renz, D.; Schaper, W.; Karliczek, G.F. Hypoxia induces permeability in brain microvessel endothelial cells via VEGF and NO. Am. J. Physiol. 1999, 276, C812–C820. [Google Scholar] [CrossRef]
  39. Mark, K.S.; Davis, T.P. Cerebral microvascular changes in permeability and tight junctions induced by hypoxia-reoxygenation. Am. J. Physiol. Heart Circ. Physiol. 2002, 282, H1485–H1494. [Google Scholar] [CrossRef] [Green Version]
  40. Cipolla, M.J.; Crete, R.; Vitullo, L.; Rix, R.D. Transcellular transport as a mechanism of blood-brain barrier disruption during stroke. Front. Biosci. 2004, 9, 777–785. [Google Scholar] [CrossRef] [Green Version]
  41. Witt, K.A.; Mark, K.S.; Huber, J.; Davis, T.P. Hypoxia-inducible factor and nuclear factor kappa-B activation in blood-brain barrier endothelium under hypoxic/reoxygenation stress. J. Neurochem. 2005, 92, 203–214. [Google Scholar] [CrossRef]
  42. Yang, F.; Zhang, X.; Sun, Y.; Wang, B.; Zhou, C.; Luo, Y.; Ge, P. Ischemic postconditioning decreases cerebral edema and brain blood barrier disruption caused by relief of carotid stenosis in a rat model of cerebral hypoperfusion. PLoS ONE 2013, 8, e57869. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Liu, X.-R.; Luo, M.; Yan, F.; Zhang, C.-C.; Li, S.-J.; Zhao, H.-P.; Ji, X.-M.; Luo, Y.-M. Ischemic postconditioning diminishes matrix metalloproteinase 9 expression and attenuates loss of the extracellular matrix proteins in rats following middle cerebral artery occlusion and reperfusion. CNS Neurosci. Ther. 2012, 18, 855–863. [Google Scholar] [CrossRef] [PubMed]
  44. Lukic-Panin, V.; Deguchi, K.; Yamashita, T.; Shang, J.; Zhang, X.; Tian, F.; Liu, N.; Kawai, H.; Matsuura, T.; Abe, K. Free radical scavenger edaravone administration protects against tissue plasminogen activator induced oxidative stress and blood brain barrier damage. Curr. Neurovasc. Res. 2010, 7, 319–329. [Google Scholar] [CrossRef]
  45. Yang, Y.; Estrada, E.Y.; Thompson, J.F.; Liu, W.; Rosenberg, G.A. Matrix metalloproteinase-mediated disruption of tight junction proteins in cerebral vessels is reversed by synthetic matrix metalloproteinase inhibitor in focal ischemia in rat. J. Cereb. Blood Flow Metab. 2007, 27, 697–709. [Google Scholar] [CrossRef] [PubMed]
  46. Giebel, S.J.; Menicucci, G.; McGuire, P.G.; Das, A. Matrix metalloproteinases in early diabetic retinopathy and their role in alteration of the blood-retinal barrier. Lab. Investig. 2005, 85, 597–607. [Google Scholar] [CrossRef]
  47. Wunderlich, M.T.; Ebert, A.D.; Kratz, T.; Goertler, M.; Jost, S.; Herrmann, M. Early neurobehavioral outcome after stroke is related to release of neurobiochemical markers of brain damage. Stroke 1999, 30, 1190–1195. [Google Scholar] [CrossRef]
  48. Connolly, E.S.; Winfree, C.J.; Rampersad, A.; Sharma, R.; Mack, W.J.; Mocco, J.; Yoshida, K.; Fujiwara, S.; Terasaki, K.; Kawamura, T.; et al. Serum S100B protein levels are correlated with subclinical neurocognitive declines after carotid endarterectomy. Neurosurgery 2001, 49, 1076–1083. [Google Scholar]
  49. Brightwell, R.; Sherwood, R.; Athanasiou, T.; Hamady, M.; Cheshire, N.J.W. The Neurological Morbidity of Carotid Revascularisation: Using Markers of Cellular Brain Injury to Compare CEA and CAS. Eur. J. Vasc. Endovasc. Surg. 2007, 34, 552–560. [Google Scholar] [CrossRef] [Green Version]
  50. Capoccia, L.; Speziale, F.; Gazzetti, M.; Mariani, P.; Rizzo, A.; Mansour, W.; Sbarigia, E.; Fiorani, P. Comparative study on carotid revascularization (endarterectomy vs. stenting) using markers of cellular brain injury, neuropsychometric tests, and diffusion-weighted magnetic resonance imaging. J. Vasc. Surg. 2010, 51, 584–591. [Google Scholar] [CrossRef] [Green Version]
  51. Solenov, E.; Watanabe, H.; Manley, G.T.; Verkman, A.S. Sevenfold-reduced osmotic water permeability in primary astrocyte cultures from AQP-4-deficient mice, measured by a fluorescence quenching method. Am. J. Physiol. Cell. Physiol. 2004, 286, C426–C432. [Google Scholar] [CrossRef]
  52. Amiry-Moghaddam, M.; Frydenlund, D.S.; Ottersen, O.P. Anchoring of aquaporin-4 in brain: Molecular mechanisms and implications for the physiology and pathophysiology of water transport. Neuroscience 2004, 129, 999–1010. [Google Scholar] [CrossRef] [PubMed]
  53. Papadopoulos, M.C.; Manley, G.T.; Krishna, S.; Verkman, A.S. Aquaporin-4 facilitates reabsorption of excess fluid in vasogenic brain edema. FASEB J. 2004, 18, 1291–1293. [Google Scholar] [CrossRef] [PubMed]
  54. Verkman, A.S.; Binder, D.K.; Bloch, O.; Auguste, K.; Papadopoulos, M.C. Three distinct roles of aquaporin-4 in brain functionrevealed by knockout mice. Biochim. Biophys. Acta 2006, 1758, 1085–1093. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  55. Higashida, T.; Kreipke, C.W.; Rafols, J.A.; Peng, C.; Schafer, S.; Schafer, P.; Ding, J.Y.; Dornbos, D.; Li, X.; Guthikonda, M.; et al. The role of hypoxia-inducible factor-1alpha, aquaporin-4, and matrix metalloproteinase-9 in blood–brain barrier disruption and brain edema after traumatic brain injury. J. Neurosurg. 2011, 114, 92–101. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  56. Davies, D.C. Blood–brain barrie breakdown In septic encephalopathy and brain tumours. J. Anat. 2002, 200, 639–646. [Google Scholar] [CrossRef]
  57. Manley, G.T.; Binder, D.K.; Papadopoulos, M.C.; Verkman, A.S. New insights into water transport and edema in the centralnervous system from phenotype analysis of quaporin-4 null mice. Neuroscience 2004, 129, 983–991. [Google Scholar] [CrossRef] [PubMed]
Ijerph 19 06210 g001 550
Figure 1. MMP-2, 72 kDa and MMP-9, 92 kDa in the next stages of research (1, 24 h before surgery; 2, 8 h post-surgery; 3, 24 h post-surgery; and 4, 48 h post-surgery).
Figure 1. MMP-2, 72 kDa and MMP-9, 92 kDa in the next stages of research (1, 24 h before surgery; 2, 8 h post-surgery; 3, 24 h post-surgery; and 4, 48 h post-surgery).
Ijerph 19 06210 g001
Ijerph 19 06210 g002 550
Figure 2. Metalloproteinase-2 activity in patients in the next stages of research (1, 24 h before surgery; 2, 8 h post-surgery; 3, 24 h post-surgery; and 4, 48 h post-surgery).
Figure 2. Metalloproteinase-2 activity in patients in the next stages of research (1, 24 h before surgery; 2, 8 h post-surgery; 3, 24 h post-surgery; and 4, 48 h post-surgery).
Ijerph 19 06210 g002
Ijerph 19 06210 g003 550
Figure 3. Metalloproteinase-9 activity in patients in the next stages of research (1, 24 h before surgery; 2, 8 h post-surgery; 3, 24 h post-surgery; and 4, 48 h post-surgery).
Figure 3. Metalloproteinase-9 activity in patients in the next stages of research (1, 24 h before surgery; 2, 8 h post-surgery; 3, 24 h post-surgery; and 4, 48 h post-surgery).
Ijerph 19 06210 g003
Ijerph 19 06210 g004 550
Figure 4. Changes of MMP-2 activity immediately after CEA/CAS procedure.
Figure 4. Changes of MMP-2 activity immediately after CEA/CAS procedure.
Ijerph 19 06210 g004
Ijerph 19 06210 g005 550
Figure 5. Claudin-1 concentration in patients in the next stages of research (1, 24 h before surgery; 2, 8 h post-surgery; 3, 24 h post-surgery; and 4, 48 h post-surgery).
Figure 5. Claudin-1 concentration in patients in the next stages of research (1, 24 h before surgery; 2, 8 h post-surgery; 3, 24 h post-surgery; and 4, 48 h post-surgery).
Ijerph 19 06210 g005
Ijerph 19 06210 g006 550
Figure 6. Occludin concentration in patients in the next stages of research (1, 24 h before surgery; 2, 8 h post-surgery; 3, 24 h post-surgery; and 4, 48 h post-surgery).
Figure 6. Occludin concentration in patients in the next stages of research (1, 24 h before surgery; 2, 8 h post-surgery; 3, 24 h post-surgery; and 4, 48 h post-surgery).
Ijerph 19 06210 g006
Ijerph 19 06210 g007 550
Figure 7. Changes occludin activity in 1st postoperative day after CEA/CAS procedure.
Figure 7. Changes occludin activity in 1st postoperative day after CEA/CAS procedure.
Ijerph 19 06210 g007
Ijerph 19 06210 g008 550
Figure 8. Aquaporin-4 concentration in patients in the next stages of research (1, 24 h before surgery; 2, 8 h post-surgery; 3, 24 h post-surgery; and 4, 48 h post-surgery).
Figure 8. Aquaporin-4 concentration in patients in the next stages of research (1, 24 h before surgery; 2, 8 h post-surgery; 3, 24 h post-surgery; and 4, 48 h post-surgery).
Ijerph 19 06210 g008
Ijerph 19 06210 g009 550
Figure 9. S100β protein concentration in patients in the next stages of research (1, 24 h before surgery; 2, 8 h post-surgery; 3, 24 h post-surgery; and 4, 48 h post-surgery).
Figure 9. S100β protein concentration in patients in the next stages of research (1, 24 h before surgery; 2, 8 h post-surgery; 3, 24 h post-surgery; and 4, 48 h post-surgery).
Ijerph 19 06210 g009
Table
Table 1. Characteristics of parameters in patients.
Table 1. Characteristics of parameters in patients.
Numer of Patients%
SexFemale2123.9
Male6776.1
Right carotid artery stenosisAbsent4450.0
Present4450.0
Left carotid artery stenosisAbsent1921.6
Present6978.4
SymptomsStroke in anamnesis2933.0
TIA in anamnesis2022.7
No symptoms3944.3
Operated sideRight3337.5
Left5562.5
Type of surgeryCEA6675.0
CAS2225.0
Plaque stability post-surgeryYes7079.5
No1820.5
Table
Table 2. Comparative analysis of the concentration of the S100β protein according to the type of surgery.
Table 2. Comparative analysis of the concentration of the S100β protein according to the type of surgery.
Stages of ResearchSum.Rang CEASum.Rang CASUZn CEAn CASStatistical Significance
12694.001222.00483−2.336622p = 0.02
22661.501254.50450−2.656622p = 0.01
32791.501124.50580−1.406622p = 0.16
42781.001135.00570−1.506622p = 0.13
Table
Table 3. Analysis of claudin-1, occludin, aquaporin-4, S100β protein, MMP-4, and MMP-9 concentrations according to the stability of the atherosclerotic plaque (S—stable plaque, U—unstable plaque).
Table 3. Analysis of claudin-1, occludin, aquaporin-4, S100β protein, MMP-4, and MMP-9 concentrations according to the stability of the atherosclerotic plaque (S—stable plaque, U—unstable plaque).
Stages of ResearchPlaqueMedianInterquartile RangeStatistical Significance
Claudin-1 (ng/mL)1S0.050.03–0.08p = 0.73
U0.060.02–0.11
2S0.060.03–0.16p = 0.56
U0.060.02–0.14
3S0.090.03–0.20p = 0.97
U0.090.03–0.26
4S0.130.07–0.29p = 0.23
U0.110.06–0.17
Occludin (ng/mL)1S0.950.81–1.36p = 0.41
U1.050.94–1.29
2S0.880.7–1.18p = 0.61
U0.920.75–1.35
3S0.940.7–1.34p = 0.49
U1.050.83–1.26
4S1.050.81–1.36p = 0.31
U1.10.94–1.45
Aquaporin-4 (U/L)1S7.985.11–10.59p = 0.85
U8.673.92–11.85
2S7.464.31–14.29p = 0.38
U12.854.24–16.9
3S7.114.37–13.96p = 0.19
U10.695.28–15.55
4S10.985.5–15.76p = 0.2
U15.866.37–20.6
S100β protein (ng/mL)1S36.2413.35–119.7p = 0.02 *
U4.982.06–105.8
2S26.826.4–108.49p = 0.07
U6.493.43–32.3
3S109.3959.28–146.54p = 0.95
U112.0826.96–153.74
4S117.9433.1–169.48p = 0.26
U63.374.67–148.6
MMP-2 (U/L)1S27,385.831475.42–109,346.2p = 0.89
U26,377.891495.89–72,648.7
2S18,398.3368.23–78,037.9p = 0.72
U14,402.55428.91–72,379.6
3S20,191.14919.58–73,871.1p = 0.65
U14,444.781081.33–73,571.4
4S23,236.04543.34–70,777.9p = 0.68
U21,306.021818.70–65,705.8
MMP-9 (U/L)1S8577.031702.92–163,367.7p = 0.05
U18,161.18561.46–201,649.1
2S18,856.581083.04–210,380.5p = 0.13
U43,023.424508.04–135,847.3
3S15,813.51314.43–200,731.2p = 0.36
U18,692.941367.15–227,834.6
4S7932.61396.33–138,148.9p = 0.1
U24,738.392729.86–193,071.7
* Statistical significance.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Terlecki, P.; Przywara, S.; Terlecki, K.; Janczak, D.; Antkiewicz, M.; Zubilewicz, T. Effect of Reconstructive Procedures of the Extracranial Segment of the Carotid Arteries on Damage to the Blood–Brain Barrier. Int. J. Environ. Res. Public Health 2022, 19, 6210. https://doi.org/10.3390/ijerph19106210

AMA Style

Terlecki P, Przywara S, Terlecki K, Janczak D, Antkiewicz M, Zubilewicz T. Effect of Reconstructive Procedures of the Extracranial Segment of the Carotid Arteries on Damage to the Blood–Brain Barrier. International Journal of Environmental Research and Public Health. 2022; 19(10):6210. https://doi.org/10.3390/ijerph19106210

Chicago/Turabian Style

Terlecki, Piotr, Stanisław Przywara, Karol Terlecki, Dariusz Janczak, Maciej Antkiewicz, and Tomasz Zubilewicz. 2022. "Effect of Reconstructive Procedures of the Extracranial Segment of the Carotid Arteries on Damage to the Blood–Brain Barrier" International Journal of Environmental Research and Public Health 19, no. 10: 6210. https://doi.org/10.3390/ijerph19106210

APA Style

Terlecki, P., Przywara, S., Terlecki, K., Janczak, D., Antkiewicz, M., & Zubilewicz, T. (2022). Effect of Reconstructive Procedures of the Extracranial Segment of the Carotid Arteries on Damage to the Blood–Brain Barrier. International Journal of Environmental Research and Public Health, 19(10), 6210. https://doi.org/10.3390/ijerph19106210

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop