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Correlation of Homocysteine with Cerebral Hemodynamic Abnormality, Endothelial Dysfunction Markers, and Cognition Impairment in Patients with Traumatic Brain Injury
Accepted Manuscript Correlation of Homocysteine with Cerebral Hemodynamic Abnormality, Endothelial Dysfunction Markers and Cognition Impairment in Patients with Traumatic Brain Injury Masoud Hatefi, Someyeh Behzadi, Masoud Moghadas Dastjerdi, Alireza Abootalebi Ghahnavieh, Asghar Rahmani, Fatemeh Madizadeh, Mohammad Reza Hafezi Ahmadi, Khairollah Asadollahi PII:
S1878-8750(16)30910-X
DOI:
10.1016/j.wneu.2016.09.080
Reference:
WNEU 4620
To appear in:
World Neurosurgery
Received Date: 27 June 2016 Revised Date:
16 September 2016
Accepted Date: 20 September 2016
Please cite this article as: Hatefi M, Behzadi S, Dastjerdi MM, Ghahnavieh AA, Rahmani A, Madizadeh F, Hafezi Ahmadi MR, Asadollahi K, Correlation of Homocysteine with Cerebral Hemodynamic Abnormality, Endothelial Dysfunction Markers and Cognition Impairment in Patients with Traumatic Brain Injury, World Neurosurgery (2016), doi: 10.1016/j.wneu.2016.09.080. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Correlation of Homocysteine with Cerebral Hemodynamic Abnormality, Endothelial Dysfunction Markers and Cognition Impairment in Patients with Traumatic Brain Injury
Masoud Hatefi1, Someyeh Behzadi2, , Masoud Moghadas Dastjerdi3, Alireza Abootalebi Ghahnavieh4, Asghar Rahmani5, Fatemeh Madizadeh6, Mohammad Reza Hafezi Ahmadi7, Khairollah Asadollahi8
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1. Department of Neurosurgery, Faculty of Medicine, Ilam University of medical sciences, Ilam, Iran. 2. Department of Radiology, Faculty of Medicine, Ilam University of medical sciences, Ilam, Iran 3. Department of Emergency Medicine, Faculty of Medicine, Isfahan University of Medical sciences, Isfahan, Iran 4. Department of Emergency Medicine, Faculty of Medicine, Isfahan University of Medical sciences, Isfahan, Iran 5. Medical student, Student researches committee, Faculty of Medicine, Ilam University of Medical Sciences, and Ilam, Iran 6. Department of Emergency Medicine, Faculty of Medicine, Ilam University of Medical sciences, Ilam, Iran 7. Department of Pathology, Faculty of Medicine, Ilam University of Medical sciences, Ilam, Iran. 8- Department of Social Medicine, Faculty of Medicine, Ilam University of Medical sciences, Ilam, Iran
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Corresponding Author: Dr Mohammad Reza Hafezi. Dept. of Pathology, Faculty of Medicine, Ilam University of Medical Sciences, Ilam, Iran, Email: [email protected] Tel: +989123765468 Fax: +988412227120
Running Title: Level of Homocysteine in Traumatic Brain Injury
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Abstract Objective: This study aimed to assess any correlation between serum levels of homocysteine (Hcy) with markers of cerebral hemodynamic, endothelial dysfunction and cognition impairment
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in patients with traumatic brain injury (TBI).
Methods: By a cross-sectional study, all clinical data and serum levels of homocysteine of 85 TBI patients were collected. The pulsatility indices (PI) of the middle cerebral artery (MCA) were recorded by transcranial color-coded doppler ultrasonography (TCD) and cereberovascular
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reactivity (CVR). Serum levels of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1), cognition status by Montreal Cognitive Assessment (MCA)
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and Mini-Mental State Examination (MMSE) were measured in all participants.
Results: Totally 85 patients including 51.76% male and the mean age of 54.48 years were studied. Level of Hcy in patients who died in hospital or during six months after TBI was significantly higher than survived patients (p=0.045, p=0.020, respectively). Also the levels of ICAM-1, VCAM-1 and PI in deceased patients were higher than their figures in survived
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individuals in both hospital and six months follow up (p=0.450, p=0.000; p=0.072, p=0.000 and p=0.090, p=0.000, respectively). CVR in deceased patients was significantly lower than that in alive individuals (p=0.008 and p=0.000, respectively). A significant correlation was found between Hcy with cognition impairment according to MCA, MMSE and cerebral hemodynamic
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status according to PI (p=0.000 for all). Also this correlation was shown between Hcy with
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ICAM-1 and VCAM-1 in hospital and six months follow up ( p=0.000 for both).
Conclusion: Hcy has a significant correlation with markers of cerebrovascular, endothelial and cognition abnormality in TBI patients. Key Words: Traumatic Brain Injury, Homocysteine, Cerebral Hemodynamic, Endothelial Dysfunction, Cognition Impairment
Introduction
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TBI is a major public health concern worldwide that is associated with high prevalence of mortality, morbidity and many socio-economical consequences in the community (1). A variety of factors such as etiology, clinical presentation and neuropathology mechanism determine the prognosis of TBI patients, and therefore outcome prediction of TBI patients is very difficult (2).
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One of the major problems in traumatic patients is their neuropsychology consequences, which impress the life's quality of patients seriously. This event can include personality changes, learning and memorial dysfunction, motor of verbal impairments and epilepsy (3). Actually, cognition disorders among TBI patients can lead to more disabilities than their physical
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dysfunctions (4). Some studies have reported the neurocognitive deficiency associated with TBI (5). In recent years, non-invasive methods for assessment of brain hemodynamic such as TCD
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were introduced and a variety of cerebral hemodynamic parameters including PI could be shown (6). One of the mechanisms associated with cognitive impairment is abnormality of cerebral hemodynamic parameters, such as CVR. Although risk factors for hemodynamic abnormalities have been found in different conditions such as cardiovascular diseases and diabetes (7) or neurodegenerative diseases such as Alzheimer's and Parkinson (8). So far, few studies were performed to determine biomarkers or risk factors related to hemodynamic changes in patients In TBI patients, secondary neurological damages lead to initiate a series of
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with TBI.
inflammatory and oxidative processes, changes in CBF, intracranial damages and diffuse axonal injuries (DAI) (9). Homocysteine (Hcy) is derived from methionine and is an intermediate sulfhydryl-containing amino acid. High level of Hcy is associated with various deleterious and
chronic
diseases
such
as
cardiovascular,
cereberovascular
and
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manifestations
neurodegenerative diseases (10). Also epidemiological studies showed that Hcy is a potent
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biomarker of pathophysiology of many neuropsychiatric disorders and cognition impairments (11). Hyperhomocysteinemia (hHcy) is a biomarker of neurotoxicity, apoptosis and leads to brain damages in both in vivo and in vitro studies. Oxidative and inflammatory processes induced by Hcy can significantly create systemic and structural brain damages (12). Some mechanisms of brain damages induced by hHcy included oxidative stress, structural and functional abnormalities of CBF, production of low-density lipoprotein, neural cell apoptosis in hippocampus, pro-atherogenic and pro-thrombotic effects (13). Correlation between variants of the endothelial nitric oxide (NO) gene and CBF after severe traumatic brain injury has been reported by different studies and on the other hand, previous studies have revealed that Hcy can
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decrease the production and bioavailability of endothelial nitric oxide (Enos) and this is considered as cause of endothelial cell damage and dysfunction (14). In addition to Enos, other markers such as VCAM-1 and ICAM-1 have direct relationships with endothelial dysfunction of TBI (15).
Hcy can impair the function of cereberovascular endothelium by multiple
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mechanisms, one of which is dysfunction of Enos (16). Hcy with increased levels of cytokines in the brain tissue can cause a neuroinflammation process that causes blood-brain barrier (BBB) dysfunction and impaired vascular integrity of the brain (17). Various markers of blood-brain barrier destruction and hemodynamic abnormality have been reported in TBI patients (18), but
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the relationship between Hcy and cerebral hemodynamic parameters in traumatic patients has not been determined so far. In addition, the relationship between Hcy and cognition impairment in
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TBI patients has not been studied. Therefore, the objective of this study was to examine the association between Hcy and hemodynamic parameters such as CVR and TCD, cognitive impairment and markers of endothelial dysfunction in patients with TBI. Methods Study Protocol
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This study included 85 TBI patients admitted to the Emergency department of Imam Khomeini hospital in Ilam city in west of Iran during March and April 2015. The hospital is a referral and educational center in West of Iran which covers about half million population. This study was approved by the Ethics Committee of Ilam University of Medical Sciences, Iran. All participants
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or their representatives completed written informed consent forms. All patients referred to the Emergency department, were under continuous observation and physical examination including
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measuring the Glasgow Coma Scale (GCS), blood pressure (BP), heart rate (HR), respiratory rate (RR), oxygen saturation rate (O2sat) and temperature rate. CT scan was performed urgently. If needed, primary resuscitation and fluid replacement was applied for all patients. Emergency brain CT-scan conducted for all of patients according to the protocol of Marshal and colleagues (19). Traumatic brain injury in admitted patients to Emergency department was approved by physical examination, relevant history and CT-scan. If the previous clinical symptoms or signs of patients were deteriorated or new clinical signs were observed, CT-scan was repeated for involved patients.
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Inclusion criteria for patients were: age between 18 to 65 year, GCS≤8 and evidences of TBI in CT-scan. Exclusion criteria were: patients younger than 18 or older than 65 years, pregnancy, lactation, any chronic or systemic diseases such as cardiovascular, chronic heart failure, chronic kidney diseases (CKD), cereberovascular, neurodegenerative (Alzheimer's or Parkinson),
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epilepsy, diabetes, multiple trauma, injury severity score (ISS) more than 15, coagulopathy, anticoagulant treatment for hormone replacement therapy (HRT), alcoholism, drug abuse and any history of psychological problems, consumption of multivitamin or folic acid before TBI. Patients were divided into two groups based on their brain CT-scan findings: 1- diffuse injury, 2-
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focal mass lesion that was described in a previous study (20). In addition, outcomes of patients were evaluated based on mortality in hospital or six months after discharge (20).
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Laboratory Assessment
At the time of admission, 10ml of blood sample from brachial vein was collected for all participants. Samples were centrifuged for 10 minutes at 4000 rpm and then were frozen and stored at -70 ° C until the assessment time. Plasma concentration of Hyc was done on freeze samples by High Performance Liquid Chromatography (HPLC) that was described previously.
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SICAM-1 and sVCAM-1 levels were measured with sandwich enzyme immunoassay technique (Elisa) kits (Bender medical systems GmbH Viena, Austria). All interassay coefficients of variations (cv) were set on 4.1/4.7 for ICAM-1 and 3.1/5.2 for VCAM-1.
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Cognition Assessment
Cognition assessment was performed for all of patients, after 2 months of post injury. We used
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the Mini-Mental State Examination (MMSE) for assessment of cognition impairment that is an overall examination for checking of cognition status. As described by previous studies, we considered a score less than 23, out of a total score of 30, as cognitive impairment in patients with TBI (21). In addition, we used the MCA that is more sensitive than MMSE and can amend some of MMSE deficiencies. The tests include a total score of 30 and obtaining a score above 26 is considered as normal cognition status for each disease (22). All hemodynamic measurements (CVR, PI,..) were performed during the first 24 hours of admission. Cereberovascular Hemodynamic Assessment.
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We have used both transcranial doppler ultrasound and inhalation of carbon dioxide (Co2) methods for evaluation of PI. The procedure was conducted using a 2 MHz probe (EME TC 64B, Eden Medizinische Elektronik, Ueberlingen, Germany) of the temporal window angle of 30
was considered as normal and less than 1.2 as abnormal.
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degrees, 12 samples volume and a depth of 45 to 60 mm. A PI index more than or equal to 1.2
After TCD, the CVR was measured for all patients via carbon dioxide (CO2) 5% for 1-2 minutes. Arterial blood gas (ABG) examination revealed that inhalation of CO2 5% for 1 to 2
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minutes caused an increase in the arterial pressure as much as 8 mm Hg. Briefly, after 2 minutes of baseline measurements, subjects breathed a mixture of CO2 5% and air for 2 minutes. Blood pressure was measured either at rest or during CO2 inhalation. CVR was calculated as percent
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rise in the ipsilateral MCA mean blood flow velocity (MBFV) per 1 mm Hg PCO2 once the MFV curve plateaued at its highest level during the 2-minute inhalation period (23). Statistical analysis
Data were analyzed by SPSS 19 (SPSS Inc, Chicago, USA) software. The findings were expressed as mean ± SD for quantitative variables and or frequencies for qualitative variables.
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The normality of data conducted by Kolmogorov-Smirnov test. The Student’s t-test and the chisquare test were applied for comparison of results. Correlation was applied between in hospital and six months outcomes of patients with cognition status markers of endothelial dysfunction and cereberovascular hemodynamic (CVR and TCD). A level of equal or less than 0.05 was
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Results
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considered statistically significant for all variables.
Basic and clinical features of subjects Characteristics of patients are shown in Table 1. A total of 85 patients including 44 (51.76%) male and 41 (48.23%) female were studied. Mean ± standard deviation (SD) of patient's age, body mass index (BMI) and blood pressure (BP) were 54.48 ± 10.34 years, 23.54 ± 13.43 kg / m2 and 95.64 ± 20.11 mmhg, respectively. Regarding with etiology of head trauma, motor vehicle accident and motorcycle accident were the most frequent in TBI patients (25.88% and 34.11%, respectively). In addition, frequency of gunshot, falling, assault and other mechanisms
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of TBI were 11.76%, 7.05%, 11.76% and 9.41%, respectively. In the Marshall Classification according to CT scan findings, most of patients were in diffuse III class (27.05%) and frequency of diffuse I, diffuse II and IV were 18.82%, 15.29% and 20%, respectively. Also, 12.94% and 5.88% of patients included in the evacuated focal mass lesion V and focal mass lesion VI,
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respectively. Median and range of GCS in patients during admission were 5 and 3-8. Also mean ± SD days of hospitalization and days in Intensive Care Unit (ICU) were 10.82 ± 5.11 and 6.16 ± 2.76 days, respectively. In hospital outcome assessment of TBI showed that, 18 patients (21.17%) died in hospital and 67 patients (78.82%) were discharged alive from hospital. 12
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patients (14.11%) died during six months after hospital discharge and 55 patients (83.58) were still alive. According to cognition assessment of patients 2 months after discharge, the mean ±
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SD of MCA score and MMSE score were 18.91 ± 5.67 and 18.79 ± 4.86, respectively. The mean ± SD serum levels of Hcy, ICAM -1, VCAM-1, PI and CVR were 45.58 ± 27.65 µmol / l, 863.54 ± 65.75 ng / ml, 564.76 ± 33.45 ng / ml, 1.03 ± 0.24 and 3.80 ± 1.49% respectively. Correlation between Hcy levels and admission GCS and Marshall scores A relationship between homocysteine levels and admission Glasgow Coma Scale (A) as well as
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the Marshall score (B) in patients with traumatic brain injury has been shown by figure 1. Based on our findings a negative (inverse) relationship was revealed between Hcy levels and GCS scores i.e. as the level of GCS was decreased, the level of Hcy was increased. Inversely, there was a positive (direct) relationship between Hcy levels and the Marshal scores and as the level of Hcy
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was increased the Marshal scores were increased too. Correlation between Hcy with outcomes of TBI patients
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Association between Hcy, markers of endothelial or cerebral dysfunction and patients' outcomes (in hospital or six months after discharge) is shown by table 2. Mean ± SD of Hcy in patients who were discharged alive from hospital was significantly lower than deceased patients (40.84 ± 26.26 µmol/l vs. 58.27±33.48 µmol/l, p=0.045). The same results were observed in six months after discharge from hospital (43.27±26.37 µmol/l in alive vs. 62.73±12.36 µmol/l in deceased patients, p=0.020). Multivariate analysis revealed that despite the considerable effects of higher levels of Hcy on both in hospital and 6 months outcome of TBI patients compared to the lower levels of Hcy, their differences were not statistically significant (Wilks’ λ = 0.90, F (4, 124.000)
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= 1.67, p <0.16, and Wilks’ λ = 0.97, F (2, 64.000) = 1.01, p <0.37 respectively) (figure2). Correlation between markers of endothelial dysfunction and outcome of TBI patients Although, the mean ±SD of ICAM-1 and VCAM-1 in deceased patients were higher than these
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figures in alive patients during hospitalization, but their differences were not statistically significant (361.09±11.11ng/ml vs. 352.80±10.72 ng/ml, p=0.450 and 576.45±12.75 ng/ml vs. 562.42±24.69, p=0.072, respectively). However, assessment of these markers in six months after discharge from hospital showed that, the levels of ICAM -1 and VCAM-1 in decesead patients
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were significanlty higher than that amng those with alive outcomes (398.27±13.64 ng/ml vs. 352.71±10.64, p=0.000 and 603.55±17.31 ng/ml vs. 562.63±24.52 ng/ml, p=0.000, respectively).
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Correlation between markers of cerebral hemodynamic dysfunction and outcome of TBI patients
Evaluation of cerebral hemodynemic parameters showed that, the mean±SD of PI in patients with decesead outcome during hospitalisation (1.16±0.30) was insignificantly higher than that in alive patients (1.00±0.28, p=0.092). However, assessment of this marker in six months after discharge from hospital showed a higher level among deceased patients (1.71±0.78) compared to
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survivors (0.99±0.28) with a significant difference (p=0.000). Also, mean ±SD of CVR in alive patients during hospitalisation and six months after discharge was significanlty higher than decesead patients (4.02±1.44 % vs. 2.73±1.27%, p=0.008, and 4.04±1.43% vs. 2.15±1.23%,
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p=0.000, respectively).
Correlation between markers of cognition status and outcome of TBI patients
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The mean ± SD of MCA score in patients with decesead outcome, six months after discharge, was significantly lower than that in alive patients (13.43±3.33 vs. 19.77±5.83, p=0.021, respectively). The same result was revealed for MMSE score, six months after discharge, (12.76±5.32 vs. 19.52±4.81, p=0.026, respectively). Correlation between Hcy and cognition status or cerebral hemodynamic abnormality Relationship between Hcy and cognition status, 2 months after TBI, is shown by fiugre 3. Based on the results of this study, the mean ± SD of Hcy in patients with MCA abnormal scores was significantly higher than that in patients with MCA normal scores ( 55.63±10.43 µmol/l vs.
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16.75±10.45 µmol/l, p=0.000, respectively). The level of Hcy in TBI patients, according to MMSE score, is shown by figure 4. Simillar to MCA score, the mean ±SD of Hcy in patients with MMSE abnormal scores was significantly higher than that in patients with MMSE normal scores (58.11±7.43 µmol/l vs 20.54±10.43 µmol/l, p=0.000). The assocaition between level of
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Hcy and PI in TBI patients is shown by figure 5. Mean ± SD of Hcy in patients with abnormal PI was significantly higher than that in those with normal PI (63.59±15.02 µmol/l vs. 33.1µmol/l , p=0.000).
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Discussion
Initial assessment of severity of head injury is one of the important components for determination
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of secondary neuropsychological prognosis in TBI patients and therapeutic decisions based on the different clinical conditions of patients. Many studies have reported that traditional methods for initial assessment of TBI patients such as CT scan or cognition parameters such as GCS are limited in determining the prognosis of patients and cannot give enough and detailed information about the secondary neurological damages (24). Accordingly, in recent years researchers are trying to find some specific markers associated with cellular and molecular neuronal injury to
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determine, faster and better, the prognosis of TBI patients (25). In this regard, we investigated the correlation between Hcy and markers related to cerebral hemodynamic abnormality such as PI and CVR, and markers of endothelial dysfunction such as ICAM-1 and VCAM-1 and cognition assessment parameters such as MCA and MMSE scores. Also we analyzed the correlation of these
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parameters with outcome of TBI patients according to in hospital stay and six months after discharge. Totally, we analyzed data of 85 TBI patients with a mean GCS of 7.25 and most of
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patients were categorized in diffuse III class (27.05%) and their lowest rate were categorized in focal mass lesion VI (5.88%), according to the Marshall classification (26). Mean ± SD of Hcy in our study was 45.58 ± 27.65 µmol/l that was significantly higher than that in healthy individuals (26). So far, only one study has measured Hcy levels in TBI patients and reported that the level of Hcy was significantly correlated with the severity of head injury, according to CT findings in Marshall Classification and GCS, in TBI patients (27). We have focused, in this study, on the relationship between Hcy with cerebral hemodynamic abnormality endothelial dysfunction markers and cognition assessment discharge in TBI patients and also correlation of this parameters with outcome of TBI patients. All patients with deceased outcomes either in hospital or
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six months after discharge had a significantly higher level of Hcy than patients with alive outcomes. In addition, the levels of ICAM-1 and VCAM-1 markers in patients with deceased outcome in hospital and six months later were higher than these figures in patients with survival outcome, but only significant among those evaluated six months after discharge from hospital.
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Previous studies showed that ICAM-1 expression caused the brain leukocyte accumulation and leukocyte-mediated tissue injury especially in CNS (15). Also animal study showed that inhibition of ICAM-1 expression led to decrease of neurological function and brain tissue in TBI patients by effect on activation of cerebral endothelial inflammatory cascade (15). So far, no study was
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conducted on the relationship assessment of ICAM-1 or VCAM-1 and outcomes in TBI patients and our study is the first report of this issue. Regarding with cerebral hemodynamic abnormality,
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the mean of PI in patients with survival outcome, either in hospital or six months later, was lower than that in deceased patients. These results are conformed to the findings of other studies, so that Jaffres and colleagues reported that high level of PI was associated with the secondary neurological deterioration in patients with mild to moderate TBI (28). The former study analyzed only prognosis of TBI during 7 days after trauma, while our study assessed the outcome of patients both in hospital and in six months after discharge as well as the relationship between PI
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and six months outcome in TBI patients. The mean of CVR in patients with survival outcome was significantly higher than that in deceased patients either in hospital or six months later. Recent studies have shown that CVR was significantly correlated with intracranial pressure (ICP) and arterial blood pressure (ABP) in TBI patients (29). We showed that the mean of MCA and MMSE
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scores in deceased patients were significantly lower than them in patients with survival outcome, six months after discharge. Other studies have shown that the cognition status was significantly
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associated with outcome of TBI patients (30). We analyzed the correlation between Hcy and markers of cerebral hemodynamic and cognition abnormality in TBI patients. Based on our findings, the level of Hcy in patients with abnormal MCA and MMSE scores was significantly higher than that in patients with normal scores. Several, case-control studies have reported that patients with cerebrovascular diseases experienced increased levels of plasma homocysteine (31, 33). Hence, Hcy could be considered as a risk factor for ischemic stroke among general population. The association between increased levels of Hcy and the incidence of cerebral ischemia has not been elucidated so far, and this phenomenon may be related to well-known ability of Hcy to induce vascular damage, atherosclerosis, and thrombosis (32). Also,
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homocysteine impairs cerebrovascular regulation which can effect on the ability of the brain circulation to compensate the blood flre reduction during ischemia. Hcy could compromise vascular regulation by interfering with the action of endothelial vasodilators such as NO and it can also impair compensatory vasodilatory mechanisms and worsen the outcome of cerebral ischemia.
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This hypothesis was proposed after a vascular reactivity to ACh, most likely mediated by release of endothelial NO, which was reduced in hyperhomocysteinemia (34). Another possible
mechanism for the vascular effects of homocysteine was autoxidation of the thiol group, a reaction
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that produces the reactive oxygen species superoxide anion (33).
It has been reported that Hcy reduces nitric oxide (NO) bioavailability by stimulating the formation of reactive oxygen species (34) and increasing matrix metalloproteinase activity,
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which results in alteration of vascular elastin/collagen ratio and compliance reduction of arterial walls (35) and it eventually causes a reduction of vessel radius by thickening arterial wall (16). A significant association between plasma total Homocysteine (tHcy) and increased vascular outcomes was reported previously (16). A relationship between Hcy and cerebrovascular indices has been reported by cross-sectional studies. For example, Lim and colleagues reported a significant relationship between Hcy levels and PI index among patients with ischemic stroke
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(36). In addition, Zhang et al showed that Hcy, at the presence of Cu21, reduces resting cerebral blood flow and attenuates the increases in CBF produced by ACh, hypercapnia, and S-nitroso-Nacetylpenicillamine responses that depend on NO (37). In general, according to different reports,
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a logical relationship between Hcy levels and characteristics of brain vessels either in asymptomatic individuals or patients in different clinical status could be considered. Our study was the first report to investigate the relationship between Hcy and cereberovascular activity
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parameters as well as cognitive status of TBI patients.An inverse association between Hcy level and cognition impairments upon the scales such as MMSE was reported in recent studies (38). The relationship between hHcy and cognitive impairments was related to some conditions such as stroke, Alzheimer's, Parkinson, epilepsy, depression and vascular dementia (11) and, in this study for the first time, we reported a relationship between hHcy and cognitive impairments in TBI patients. In addition, we showed that, there is a significant relationship between Hcy and cerebral hemodynamic abnormality according to PI in TBI patients as the first report in this field. PI is one of the most valuable indicators of early hemodynamic changes obtained by TCD that is
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a good indicator of cerebral vascular resistance (39). Recent studies have reported a direct correlation between PI and prognosis of patients with TBI (40) that was in accordance with our findings. The relationship between Hcy and PI can be linked with other effects of Hcy on the CNS, so that, Hcy by disrupting the function of NO (14) can be involved in the regulation of
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TBI-induced dysregulation of CBF. Of course, this is only a hypothesis and there is no relevant evidence so far in the literature. Furthermore, high level of Hcy has a direct association with up regulation of matrix metalloproteinase’s (MMPs), membrane proteins including tight junctions (TJs) and dysfunction and reduction of endothelial cells forming blood brain barrier (BBB) (34).
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All of these mechanisms can explain the role of Hcy in cereberovascular toxicity (37, 6). We showed a significant correlation between Hcy and cerebral hemodynamic as well as endothelial
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dysfunction makers in TBI patients according to an assessment either in hospital or in six months after discharge. Also, Hcy has a direct association with PI, ICAM-1 and VCAM-1 levels according to in hospital outcome, but a reverse association with CVR in TBI patients. Based on our findings, there is an inverse relationship, between Hcy and markers of cognitive function such as MCA and MMSE six months after discharge in TBI patients. This result was also found for correlation between level of Hcy and CVR. In general, according to our results, we can say
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that Hcy has a direct correlation with outcome of TBI patients. Hcy has also a logical connection parameter with cerebral hemodynamic and endothelial dysfunction markers. The relationship between Hcy and cognitive impairments is another important finding of our study. So far, no specific study has been done in this field and this is the first report on the relationship between
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Hcy and vascular parameters and cognitive performance in TBI patients. Although our study couldn't reveal any direct relation between serum levels of Hcy and patients prognosis but based
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on our findings the levels of Hcy were associated with variables such as GCS, Marshal, CVR and PI that effect on patients' prognosis. Therefore, a possible relation between the levels of Hcy and TBI patients' prognosis could be suggested by our study. This issue requires deep investigations in the field of Hcy in TBI patients. Since the nitrite oxide is one of the markers associated with cereberovascular outoregulation system (42) and its measurement can help to understand the role of Hcy in this system, one of limitations of our study was lack of measurement of NO, and we hope it could be resolved in the future studies. Conflict of interests
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The authors do not have any actual or potential conflict of interests in this study. Acknowledgements
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We thank colleagues in the Faculty of Medicine, Ilam University of Medical Sciences, participants, coordinators and data reviewers who assisted in this study. Funding/Support
Ilam University of Medical Sciences, Ilam, Iran, supported this study. Authors’ contributions
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Hatefi M and Rahmani A and contributed to data analysis, interpretation of the results and drafted the manuscript. Rahmani A participated in the manuscript preparation. Behzadi S participated in radiology
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procedure (TCD, CT-Scan, PI and CVR assessment). Rahmani A, Masoud Dastjerdi MM, Ghahnavieh AA and Madizadeh F participated in data collection. Hatefi M and Hafezi Ahmadi MR contributed to the design of the study and Hafezi Ahmadi MR participated in the laboratory work and manuscript preparation. Hafezi Ahmadi MR and Asadollahi K participated in the manuscript preparation and critical
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review of the paper. All authors have read and approved the article for publication.
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Table 1. Demographic and clinical characteristic of the TBI patients.
29(34.11) 22(25.88) 10(11.76) 6(7.05) 10(11.76) 8(9.41) 5(3-8)
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18(21.17) 67(78.82)
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16(18.82) 13(15.29) 23(27.05) 17(20) 11(12.94) 5(5.88) 10.82± 5.11 6.16± 2.76
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44(51.76) 41(48.23) 54.48±10.34 23.54±13.43
12(14.11) 56(83.58)
18.91±5.67 18.79±4.86 45.58±27.65 863.54±65.75 564.76±33.45 1.03±0.24 3.80±1.49
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Marshall score, n (%) Diffuse I Diffuse II Diffuse III Diffuse IV Evacuated focal mass lesion V Focal mass lesion VI Day in hospitalization Day in ICU Outcome In hospital, n (%) Deceased (GOS 1) Live (GOS 2-5) Outcome In 6 months, n (%) Deceased (GOS 1) Live (GOS 2-5) Cognition Score MCA MMSE Homocysteine (µmol/l) ICAM-1(ng/ml) VCAM-1(ng/ml) PI CVR (%)
TBI , n=85
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Characteristics Sex, n (%) Male Female Age(y) BMI( kg/m2) Injury mechanism Motor Vehicle Accident Motor Cycle Accident Gun Shot Wound Fall Assault Other GCS, median (range)
BMI; Body Mass Index, GCS; Glasgow Coma Scale, ICU; Intensive Care Units, GOS; Glasgow Outcome Scale, MCA; Montreal Cognitive Assessment, MMSE; Mini-Mental State Examination, ICAM; Intercellular Adhesion Molecule-1, VCAM-1; Vascular Cell Adhesion Molecule-1, PI; And Pulsatility Index, CVR; Cereberovascular Reactivity
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Table 2. Association between outcome of Traumatic Brain Injury and Homocysteine, endothelial and cerebral hemodynamic parameters.
58.27±33.48
P
0.045
43.27±26.37
P 0.020
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40.84±26.26
6 Months Outcomes Live Deceased 62.73±12.36
352.80±10.72 361.09±11.11 0.450
352.71±10.64 398.27±13.64 0.000
562.42±24.69 576.45±12.75 0.072
562.63±24.52 603.55±17.31 0.000
1.00±0.28 4.02±1.44 -
0.99±0.28 4.04±1.43 19.77±5.83 19.52±4.81
1.16±0.30 2.73±1.27 -
0.090 0.008 -
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Homocysteine (µmol/l) ICAM1(ng/ml) VCAM1(ng/ml) PI CVR (%) MCA Score MMSE Score
In Hospital Outcomes Live Deceased
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1.71±0.78 2.15±1.23 13.43±3.33 12.76±5.32
0.000 0.010 0.021 0.026
MCA; Montreal Cognitive Assessment, MMSE; Mini-Mental State Examination, ICAM;
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Intercellular Adhesion Molecule-1, VCAM-1; Vascular Cell Adhesion Molecule-1, PI; And Pulsatility Index, CVR; Cereberovascular Reactivity
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Figure 1. Relationship between homocysteine levels and admission Glasgow Coma Scale (A) and the Marshall score (B) in patients with traumatic brain injury 1=Diffuse I, 2=Diffuse II, 3= Diffuse III, 4=Diffuse IV, 5=Evacuated focal mass lesion V 6= Focal mass lesion VI
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A B Figure 2. Multivariate analysis of the effects of homocysteine levels on patients outcome
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A= 6 months outcome; Hcy group (0: <10 µmol/l, 1: 10-30 µmol/l, 2: >30 µmol/l); outcome (0: dead, 1: alive) . B= In hospital outcome; Hcy group (0: <10 µmol/l, 1: 10-30 µmol/l, 2: >30 µmol/l); outcome (0: dead, 1: alive)
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Homocysteine ,Mean±SD(µmol/I)
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MCA MCA Normal Abnormal
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MMSE MMSE Normal Abnormal
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Figure 3. Association between Homocysteine (µmol/l) and cognition status according to Montreal Cognitive Assessment (MCA) ( A) , according to Mini-Mental State Examination (MMSE) (B) and pulsatility index (PI) (C) in patients with Traumatic Brain Injury. Data are expressed as Mean ± SD. * =p<0.05.
B
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Figure 4. Association between Homocysteine and cerebral hemodynamic such as pulsatility index (A) or cereberovascular reactivity (B) and with markers of endothelial dysfunction such as ICAM-1(C) or VCAM-1(D) according to in hospital outcome of patients with traumatic brain injury. ICAM-1; Intercellular Adhesion Molecule-1, VCAM-1; vascular cell adhesion molecule-1.
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Figure 5. Association between Homocysteine and markers of cerebral hemodynamic such as pulsatility index (A) and cereberovascular reactivity(B) , and also with markers of endothelial dysfunctions such as ICAM-1(C) and VCAM-1(D) and with cognition score such as MCA score(E) and MMSE score (F) according to six months after discharge from hospital in patients with traumatic brain injury. ICAM-1; Intercellular Adhesion Molecule-1, VCAM-1; vascular cell adhesion molecule-1, MCA; Montreal Cognitive Assessment, MMSE; Mini-Mental State Examination
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22. Liang H, You G, Liao L, Huang Y. Clinical observation of cognitive impairment after traumatic brain injury treated with acupuncture and cognitive training. Zhongguo Zhen Jiu. 2015;35(9):865-8. 23. Marshall RS1, Rundek T, Sproule DM, Fitzsimmons BF, Schwartz S, Lazar RM. Monitoring of cerebral vasodilatory capacity with transcranial Doppler carbon dioxide inhalation in patients with severe carotid artery disease. Stroke. 2003; 34(4):945-9.
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24. Maas AI, Hukkelhoven CW, Marshall LF, Steyerberg EW: Prediction of outcome in traumatic brain injury with computed tomographic characteristics: a comparison between the computed tomographic classification and combinations of computed tomographic predictors. Neurosurgery 2005, 57:1173-1182. 25. Papa L, Akinyi L, Liu MC, Pineda JA, Tepas JJ, Oli MW, Zheng W, Robinson G, Robicsek SA, Gabrielli A, Heaton SC, Hannay HJ, Demery JA, Brophy GM, Layon J, Robertson CS, Hayes RL, Wang KK: Ubiquitin C-terminal hydrolase is a novel biomarker in humans for severe traumatic brain injury. Crit Care Med 2010, 38 :138-144
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35. Mujumdar VS, Aru GM, Tyagi SC. Induction of oxidative stress by homocyst(e)ine impairs endothelial function. J Cell Biochem . 2001; 82: 491–500. 36. Lim MH1, Cho YI, Jeong SK. Homocysteine and pulsatility index of cerebral arteries. Stroke. 2009 Oct; 40(10):3216-20. doi: 10.1161/STROKEAHA.109.558403.
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37. Zhang F1, Slungaard A, Vercellotti GM, Iadecola C. Superoxide-dependent cerebrovascular effects of homocysteine. Am J Physiol. 1998 Jun; 274(6 Pt 2):R1704-11. 38. Duthie SJ, Whalley LJ, Collins AR, Leaper S, Berger K, Deary IJ, et al. Homocysteine, B vitamin status, and cognitive function in the elderly. Am J Clin Nutr 2002; 75:908-13
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39. Ract C1, Le Moigno S, Bruder N, Vigué B. Transcranial Doppler ultrasound goal-directed therapy for the early management of severe traumatic brain injury. Intensive Care Med. 2007;33(4):645-51.
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40. Moreno JA1, Mesalles E, Gener J, Tomasa A, Ley A, Roca J, Fernández-Llamazares J. Evaluating the outcome of severe head injury with transcranial Doppler ultrasonography. Neurosurg Focus. 2000;15:8(1):e8. 41. Kamat PK, Kalani A, Givvimani S, Sathnur PB, Tyagi SC, et al. Hydrogen sulfide attenuates neurodegeneration and neurovascular dysfunction induced by intracerebral-administered homocysteine in mice. Neuroscience. 2013; 252:302–19
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Level of Hcy in dead patients after TBI was significantly higher than survived patients in both hospital and six months follow up. Level of ICAM-1, VCAM-1 and PI in deceased patients was higher than survived patients in both hospital and six months follow up in TBI patients. A significant correlation was found between Hcy with cognition impairment according to MCA, A significant correlation was found between Hcy and cerebral
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MMSE in TBI patients.
hemodynamic according to PI in TBI patients. A significant correlation was shown between Hcy
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with ICAM-1 and VCAM-1 in TBI patients.
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Hcy: Homocysteine Hhcy: Hyperhomocysteinemia TBI: Traumatic Brain Injury PI: Pulsatility Indices CVR: Cereberovascular Reactivity MCA: Middle Cerebral Artery TCD: Transcranial Color-Coded Doppler Ultrasonography MCA: Montreal Cognitive Assessment MMSE: Mini-Mental State Examination ICAM-1: Intercellular Adhesion Molecule-1 VCAM-1: Vascular Adhesion Molecule-1 Enos: Endothelial Nitric Oxide CBF: Cerebral Blood Flow GCS: Glasgow Coma Scale BP: Blood Pressure HR: Heart Rate ISS: Injury Severity Score HR: Hormone Replacement Therapy CKD: Chronic Kidney Disease HPLC: High Performance Liquid Chromatography
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List of abbreviations
S1878-8750(16)30910-X
DOI:
10.1016/j.wneu.2016.09.080
Reference:
WNEU 4620
To appear in:
World Neurosurgery
Received Date: 27 June 2016 Revised Date:
16 September 2016
Accepted Date: 20 September 2016
Please cite this article as: Hatefi M, Behzadi S, Dastjerdi MM, Ghahnavieh AA, Rahmani A, Madizadeh F, Hafezi Ahmadi MR, Asadollahi K, Correlation of Homocysteine with Cerebral Hemodynamic Abnormality, Endothelial Dysfunction Markers and Cognition Impairment in Patients with Traumatic Brain Injury, World Neurosurgery (2016), doi: 10.1016/j.wneu.2016.09.080. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Correlation of Homocysteine with Cerebral Hemodynamic Abnormality, Endothelial Dysfunction Markers and Cognition Impairment in Patients with Traumatic Brain Injury
Masoud Hatefi1, Someyeh Behzadi2, , Masoud Moghadas Dastjerdi3, Alireza Abootalebi Ghahnavieh4, Asghar Rahmani5, Fatemeh Madizadeh6, Mohammad Reza Hafezi Ahmadi7, Khairollah Asadollahi8
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1. Department of Neurosurgery, Faculty of Medicine, Ilam University of medical sciences, Ilam, Iran. 2. Department of Radiology, Faculty of Medicine, Ilam University of medical sciences, Ilam, Iran 3. Department of Emergency Medicine, Faculty of Medicine, Isfahan University of Medical sciences, Isfahan, Iran 4. Department of Emergency Medicine, Faculty of Medicine, Isfahan University of Medical sciences, Isfahan, Iran 5. Medical student, Student researches committee, Faculty of Medicine, Ilam University of Medical Sciences, and Ilam, Iran 6. Department of Emergency Medicine, Faculty of Medicine, Ilam University of Medical sciences, Ilam, Iran 7. Department of Pathology, Faculty of Medicine, Ilam University of Medical sciences, Ilam, Iran. 8- Department of Social Medicine, Faculty of Medicine, Ilam University of Medical sciences, Ilam, Iran
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Corresponding Author: Dr Mohammad Reza Hafezi. Dept. of Pathology, Faculty of Medicine, Ilam University of Medical Sciences, Ilam, Iran, Email: [email protected] Tel: +989123765468 Fax: +988412227120
Running Title: Level of Homocysteine in Traumatic Brain Injury
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Abstract Objective: This study aimed to assess any correlation between serum levels of homocysteine (Hcy) with markers of cerebral hemodynamic, endothelial dysfunction and cognition impairment
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in patients with traumatic brain injury (TBI).
Methods: By a cross-sectional study, all clinical data and serum levels of homocysteine of 85 TBI patients were collected. The pulsatility indices (PI) of the middle cerebral artery (MCA) were recorded by transcranial color-coded doppler ultrasonography (TCD) and cereberovascular
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reactivity (CVR). Serum levels of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1), cognition status by Montreal Cognitive Assessment (MCA)
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and Mini-Mental State Examination (MMSE) were measured in all participants.
Results: Totally 85 patients including 51.76% male and the mean age of 54.48 years were studied. Level of Hcy in patients who died in hospital or during six months after TBI was significantly higher than survived patients (p=0.045, p=0.020, respectively). Also the levels of ICAM-1, VCAM-1 and PI in deceased patients were higher than their figures in survived
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individuals in both hospital and six months follow up (p=0.450, p=0.000; p=0.072, p=0.000 and p=0.090, p=0.000, respectively). CVR in deceased patients was significantly lower than that in alive individuals (p=0.008 and p=0.000, respectively). A significant correlation was found between Hcy with cognition impairment according to MCA, MMSE and cerebral hemodynamic
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status according to PI (p=0.000 for all). Also this correlation was shown between Hcy with
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ICAM-1 and VCAM-1 in hospital and six months follow up ( p=0.000 for both).
Conclusion: Hcy has a significant correlation with markers of cerebrovascular, endothelial and cognition abnormality in TBI patients. Key Words: Traumatic Brain Injury, Homocysteine, Cerebral Hemodynamic, Endothelial Dysfunction, Cognition Impairment
Introduction
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TBI is a major public health concern worldwide that is associated with high prevalence of mortality, morbidity and many socio-economical consequences in the community (1). A variety of factors such as etiology, clinical presentation and neuropathology mechanism determine the prognosis of TBI patients, and therefore outcome prediction of TBI patients is very difficult (2).
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One of the major problems in traumatic patients is their neuropsychology consequences, which impress the life's quality of patients seriously. This event can include personality changes, learning and memorial dysfunction, motor of verbal impairments and epilepsy (3). Actually, cognition disorders among TBI patients can lead to more disabilities than their physical
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dysfunctions (4). Some studies have reported the neurocognitive deficiency associated with TBI (5). In recent years, non-invasive methods for assessment of brain hemodynamic such as TCD
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were introduced and a variety of cerebral hemodynamic parameters including PI could be shown (6). One of the mechanisms associated with cognitive impairment is abnormality of cerebral hemodynamic parameters, such as CVR. Although risk factors for hemodynamic abnormalities have been found in different conditions such as cardiovascular diseases and diabetes (7) or neurodegenerative diseases such as Alzheimer's and Parkinson (8). So far, few studies were performed to determine biomarkers or risk factors related to hemodynamic changes in patients In TBI patients, secondary neurological damages lead to initiate a series of
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with TBI.
inflammatory and oxidative processes, changes in CBF, intracranial damages and diffuse axonal injuries (DAI) (9). Homocysteine (Hcy) is derived from methionine and is an intermediate sulfhydryl-containing amino acid. High level of Hcy is associated with various deleterious and
chronic
diseases
such
as
cardiovascular,
cereberovascular
and
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neurodegenerative diseases (10). Also epidemiological studies showed that Hcy is a potent
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biomarker of pathophysiology of many neuropsychiatric disorders and cognition impairments (11). Hyperhomocysteinemia (hHcy) is a biomarker of neurotoxicity, apoptosis and leads to brain damages in both in vivo and in vitro studies. Oxidative and inflammatory processes induced by Hcy can significantly create systemic and structural brain damages (12). Some mechanisms of brain damages induced by hHcy included oxidative stress, structural and functional abnormalities of CBF, production of low-density lipoprotein, neural cell apoptosis in hippocampus, pro-atherogenic and pro-thrombotic effects (13). Correlation between variants of the endothelial nitric oxide (NO) gene and CBF after severe traumatic brain injury has been reported by different studies and on the other hand, previous studies have revealed that Hcy can
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decrease the production and bioavailability of endothelial nitric oxide (Enos) and this is considered as cause of endothelial cell damage and dysfunction (14). In addition to Enos, other markers such as VCAM-1 and ICAM-1 have direct relationships with endothelial dysfunction of TBI (15).
Hcy can impair the function of cereberovascular endothelium by multiple
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mechanisms, one of which is dysfunction of Enos (16). Hcy with increased levels of cytokines in the brain tissue can cause a neuroinflammation process that causes blood-brain barrier (BBB) dysfunction and impaired vascular integrity of the brain (17). Various markers of blood-brain barrier destruction and hemodynamic abnormality have been reported in TBI patients (18), but
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the relationship between Hcy and cerebral hemodynamic parameters in traumatic patients has not been determined so far. In addition, the relationship between Hcy and cognition impairment in
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TBI patients has not been studied. Therefore, the objective of this study was to examine the association between Hcy and hemodynamic parameters such as CVR and TCD, cognitive impairment and markers of endothelial dysfunction in patients with TBI. Methods Study Protocol
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This study included 85 TBI patients admitted to the Emergency department of Imam Khomeini hospital in Ilam city in west of Iran during March and April 2015. The hospital is a referral and educational center in West of Iran which covers about half million population. This study was approved by the Ethics Committee of Ilam University of Medical Sciences, Iran. All participants
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or their representatives completed written informed consent forms. All patients referred to the Emergency department, were under continuous observation and physical examination including
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measuring the Glasgow Coma Scale (GCS), blood pressure (BP), heart rate (HR), respiratory rate (RR), oxygen saturation rate (O2sat) and temperature rate. CT scan was performed urgently. If needed, primary resuscitation and fluid replacement was applied for all patients. Emergency brain CT-scan conducted for all of patients according to the protocol of Marshal and colleagues (19). Traumatic brain injury in admitted patients to Emergency department was approved by physical examination, relevant history and CT-scan. If the previous clinical symptoms or signs of patients were deteriorated or new clinical signs were observed, CT-scan was repeated for involved patients.
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Inclusion criteria for patients were: age between 18 to 65 year, GCS≤8 and evidences of TBI in CT-scan. Exclusion criteria were: patients younger than 18 or older than 65 years, pregnancy, lactation, any chronic or systemic diseases such as cardiovascular, chronic heart failure, chronic kidney diseases (CKD), cereberovascular, neurodegenerative (Alzheimer's or Parkinson),
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epilepsy, diabetes, multiple trauma, injury severity score (ISS) more than 15, coagulopathy, anticoagulant treatment for hormone replacement therapy (HRT), alcoholism, drug abuse and any history of psychological problems, consumption of multivitamin or folic acid before TBI. Patients were divided into two groups based on their brain CT-scan findings: 1- diffuse injury, 2-
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focal mass lesion that was described in a previous study (20). In addition, outcomes of patients were evaluated based on mortality in hospital or six months after discharge (20).
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Laboratory Assessment
At the time of admission, 10ml of blood sample from brachial vein was collected for all participants. Samples were centrifuged for 10 minutes at 4000 rpm and then were frozen and stored at -70 ° C until the assessment time. Plasma concentration of Hyc was done on freeze samples by High Performance Liquid Chromatography (HPLC) that was described previously.
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SICAM-1 and sVCAM-1 levels were measured with sandwich enzyme immunoassay technique (Elisa) kits (Bender medical systems GmbH Viena, Austria). All interassay coefficients of variations (cv) were set on 4.1/4.7 for ICAM-1 and 3.1/5.2 for VCAM-1.
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Cognition Assessment
Cognition assessment was performed for all of patients, after 2 months of post injury. We used
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the Mini-Mental State Examination (MMSE) for assessment of cognition impairment that is an overall examination for checking of cognition status. As described by previous studies, we considered a score less than 23, out of a total score of 30, as cognitive impairment in patients with TBI (21). In addition, we used the MCA that is more sensitive than MMSE and can amend some of MMSE deficiencies. The tests include a total score of 30 and obtaining a score above 26 is considered as normal cognition status for each disease (22). All hemodynamic measurements (CVR, PI,..) were performed during the first 24 hours of admission. Cereberovascular Hemodynamic Assessment.
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We have used both transcranial doppler ultrasound and inhalation of carbon dioxide (Co2) methods for evaluation of PI. The procedure was conducted using a 2 MHz probe (EME TC 64B, Eden Medizinische Elektronik, Ueberlingen, Germany) of the temporal window angle of 30
was considered as normal and less than 1.2 as abnormal.
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degrees, 12 samples volume and a depth of 45 to 60 mm. A PI index more than or equal to 1.2
After TCD, the CVR was measured for all patients via carbon dioxide (CO2) 5% for 1-2 minutes. Arterial blood gas (ABG) examination revealed that inhalation of CO2 5% for 1 to 2
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minutes caused an increase in the arterial pressure as much as 8 mm Hg. Briefly, after 2 minutes of baseline measurements, subjects breathed a mixture of CO2 5% and air for 2 minutes. Blood pressure was measured either at rest or during CO2 inhalation. CVR was calculated as percent
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rise in the ipsilateral MCA mean blood flow velocity (MBFV) per 1 mm Hg PCO2 once the MFV curve plateaued at its highest level during the 2-minute inhalation period (23). Statistical analysis
Data were analyzed by SPSS 19 (SPSS Inc, Chicago, USA) software. The findings were expressed as mean ± SD for quantitative variables and or frequencies for qualitative variables.
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The normality of data conducted by Kolmogorov-Smirnov test. The Student’s t-test and the chisquare test were applied for comparison of results. Correlation was applied between in hospital and six months outcomes of patients with cognition status markers of endothelial dysfunction and cereberovascular hemodynamic (CVR and TCD). A level of equal or less than 0.05 was
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Results
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considered statistically significant for all variables.
Basic and clinical features of subjects Characteristics of patients are shown in Table 1. A total of 85 patients including 44 (51.76%) male and 41 (48.23%) female were studied. Mean ± standard deviation (SD) of patient's age, body mass index (BMI) and blood pressure (BP) were 54.48 ± 10.34 years, 23.54 ± 13.43 kg / m2 and 95.64 ± 20.11 mmhg, respectively. Regarding with etiology of head trauma, motor vehicle accident and motorcycle accident were the most frequent in TBI patients (25.88% and 34.11%, respectively). In addition, frequency of gunshot, falling, assault and other mechanisms
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of TBI were 11.76%, 7.05%, 11.76% and 9.41%, respectively. In the Marshall Classification according to CT scan findings, most of patients were in diffuse III class (27.05%) and frequency of diffuse I, diffuse II and IV were 18.82%, 15.29% and 20%, respectively. Also, 12.94% and 5.88% of patients included in the evacuated focal mass lesion V and focal mass lesion VI,
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respectively. Median and range of GCS in patients during admission were 5 and 3-8. Also mean ± SD days of hospitalization and days in Intensive Care Unit (ICU) were 10.82 ± 5.11 and 6.16 ± 2.76 days, respectively. In hospital outcome assessment of TBI showed that, 18 patients (21.17%) died in hospital and 67 patients (78.82%) were discharged alive from hospital. 12
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patients (14.11%) died during six months after hospital discharge and 55 patients (83.58) were still alive. According to cognition assessment of patients 2 months after discharge, the mean ±
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SD of MCA score and MMSE score were 18.91 ± 5.67 and 18.79 ± 4.86, respectively. The mean ± SD serum levels of Hcy, ICAM -1, VCAM-1, PI and CVR were 45.58 ± 27.65 µmol / l, 863.54 ± 65.75 ng / ml, 564.76 ± 33.45 ng / ml, 1.03 ± 0.24 and 3.80 ± 1.49% respectively. Correlation between Hcy levels and admission GCS and Marshall scores A relationship between homocysteine levels and admission Glasgow Coma Scale (A) as well as
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the Marshall score (B) in patients with traumatic brain injury has been shown by figure 1. Based on our findings a negative (inverse) relationship was revealed between Hcy levels and GCS scores i.e. as the level of GCS was decreased, the level of Hcy was increased. Inversely, there was a positive (direct) relationship between Hcy levels and the Marshal scores and as the level of Hcy
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was increased the Marshal scores were increased too. Correlation between Hcy with outcomes of TBI patients
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Association between Hcy, markers of endothelial or cerebral dysfunction and patients' outcomes (in hospital or six months after discharge) is shown by table 2. Mean ± SD of Hcy in patients who were discharged alive from hospital was significantly lower than deceased patients (40.84 ± 26.26 µmol/l vs. 58.27±33.48 µmol/l, p=0.045). The same results were observed in six months after discharge from hospital (43.27±26.37 µmol/l in alive vs. 62.73±12.36 µmol/l in deceased patients, p=0.020). Multivariate analysis revealed that despite the considerable effects of higher levels of Hcy on both in hospital and 6 months outcome of TBI patients compared to the lower levels of Hcy, their differences were not statistically significant (Wilks’ λ = 0.90, F (4, 124.000)
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= 1.67, p <0.16, and Wilks’ λ = 0.97, F (2, 64.000) = 1.01, p <0.37 respectively) (figure2). Correlation between markers of endothelial dysfunction and outcome of TBI patients Although, the mean ±SD of ICAM-1 and VCAM-1 in deceased patients were higher than these
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figures in alive patients during hospitalization, but their differences were not statistically significant (361.09±11.11ng/ml vs. 352.80±10.72 ng/ml, p=0.450 and 576.45±12.75 ng/ml vs. 562.42±24.69, p=0.072, respectively). However, assessment of these markers in six months after discharge from hospital showed that, the levels of ICAM -1 and VCAM-1 in decesead patients
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were significanlty higher than that amng those with alive outcomes (398.27±13.64 ng/ml vs. 352.71±10.64, p=0.000 and 603.55±17.31 ng/ml vs. 562.63±24.52 ng/ml, p=0.000, respectively).
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Correlation between markers of cerebral hemodynamic dysfunction and outcome of TBI patients
Evaluation of cerebral hemodynemic parameters showed that, the mean±SD of PI in patients with decesead outcome during hospitalisation (1.16±0.30) was insignificantly higher than that in alive patients (1.00±0.28, p=0.092). However, assessment of this marker in six months after discharge from hospital showed a higher level among deceased patients (1.71±0.78) compared to
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survivors (0.99±0.28) with a significant difference (p=0.000). Also, mean ±SD of CVR in alive patients during hospitalisation and six months after discharge was significanlty higher than decesead patients (4.02±1.44 % vs. 2.73±1.27%, p=0.008, and 4.04±1.43% vs. 2.15±1.23%,
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p=0.000, respectively).
Correlation between markers of cognition status and outcome of TBI patients
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The mean ± SD of MCA score in patients with decesead outcome, six months after discharge, was significantly lower than that in alive patients (13.43±3.33 vs. 19.77±5.83, p=0.021, respectively). The same result was revealed for MMSE score, six months after discharge, (12.76±5.32 vs. 19.52±4.81, p=0.026, respectively). Correlation between Hcy and cognition status or cerebral hemodynamic abnormality Relationship between Hcy and cognition status, 2 months after TBI, is shown by fiugre 3. Based on the results of this study, the mean ± SD of Hcy in patients with MCA abnormal scores was significantly higher than that in patients with MCA normal scores ( 55.63±10.43 µmol/l vs.
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16.75±10.45 µmol/l, p=0.000, respectively). The level of Hcy in TBI patients, according to MMSE score, is shown by figure 4. Simillar to MCA score, the mean ±SD of Hcy in patients with MMSE abnormal scores was significantly higher than that in patients with MMSE normal scores (58.11±7.43 µmol/l vs 20.54±10.43 µmol/l, p=0.000). The assocaition between level of
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Hcy and PI in TBI patients is shown by figure 5. Mean ± SD of Hcy in patients with abnormal PI was significantly higher than that in those with normal PI (63.59±15.02 µmol/l vs. 33.1µmol/l , p=0.000).
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Discussion
Initial assessment of severity of head injury is one of the important components for determination
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of secondary neuropsychological prognosis in TBI patients and therapeutic decisions based on the different clinical conditions of patients. Many studies have reported that traditional methods for initial assessment of TBI patients such as CT scan or cognition parameters such as GCS are limited in determining the prognosis of patients and cannot give enough and detailed information about the secondary neurological damages (24). Accordingly, in recent years researchers are trying to find some specific markers associated with cellular and molecular neuronal injury to
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determine, faster and better, the prognosis of TBI patients (25). In this regard, we investigated the correlation between Hcy and markers related to cerebral hemodynamic abnormality such as PI and CVR, and markers of endothelial dysfunction such as ICAM-1 and VCAM-1 and cognition assessment parameters such as MCA and MMSE scores. Also we analyzed the correlation of these
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parameters with outcome of TBI patients according to in hospital stay and six months after discharge. Totally, we analyzed data of 85 TBI patients with a mean GCS of 7.25 and most of
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patients were categorized in diffuse III class (27.05%) and their lowest rate were categorized in focal mass lesion VI (5.88%), according to the Marshall classification (26). Mean ± SD of Hcy in our study was 45.58 ± 27.65 µmol/l that was significantly higher than that in healthy individuals (26). So far, only one study has measured Hcy levels in TBI patients and reported that the level of Hcy was significantly correlated with the severity of head injury, according to CT findings in Marshall Classification and GCS, in TBI patients (27). We have focused, in this study, on the relationship between Hcy with cerebral hemodynamic abnormality endothelial dysfunction markers and cognition assessment discharge in TBI patients and also correlation of this parameters with outcome of TBI patients. All patients with deceased outcomes either in hospital or
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six months after discharge had a significantly higher level of Hcy than patients with alive outcomes. In addition, the levels of ICAM-1 and VCAM-1 markers in patients with deceased outcome in hospital and six months later were higher than these figures in patients with survival outcome, but only significant among those evaluated six months after discharge from hospital.
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Previous studies showed that ICAM-1 expression caused the brain leukocyte accumulation and leukocyte-mediated tissue injury especially in CNS (15). Also animal study showed that inhibition of ICAM-1 expression led to decrease of neurological function and brain tissue in TBI patients by effect on activation of cerebral endothelial inflammatory cascade (15). So far, no study was
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conducted on the relationship assessment of ICAM-1 or VCAM-1 and outcomes in TBI patients and our study is the first report of this issue. Regarding with cerebral hemodynamic abnormality,
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the mean of PI in patients with survival outcome, either in hospital or six months later, was lower than that in deceased patients. These results are conformed to the findings of other studies, so that Jaffres and colleagues reported that high level of PI was associated with the secondary neurological deterioration in patients with mild to moderate TBI (28). The former study analyzed only prognosis of TBI during 7 days after trauma, while our study assessed the outcome of patients both in hospital and in six months after discharge as well as the relationship between PI
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and six months outcome in TBI patients. The mean of CVR in patients with survival outcome was significantly higher than that in deceased patients either in hospital or six months later. Recent studies have shown that CVR was significantly correlated with intracranial pressure (ICP) and arterial blood pressure (ABP) in TBI patients (29). We showed that the mean of MCA and MMSE
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scores in deceased patients were significantly lower than them in patients with survival outcome, six months after discharge. Other studies have shown that the cognition status was significantly
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associated with outcome of TBI patients (30). We analyzed the correlation between Hcy and markers of cerebral hemodynamic and cognition abnormality in TBI patients. Based on our findings, the level of Hcy in patients with abnormal MCA and MMSE scores was significantly higher than that in patients with normal scores. Several, case-control studies have reported that patients with cerebrovascular diseases experienced increased levels of plasma homocysteine (31, 33). Hence, Hcy could be considered as a risk factor for ischemic stroke among general population. The association between increased levels of Hcy and the incidence of cerebral ischemia has not been elucidated so far, and this phenomenon may be related to well-known ability of Hcy to induce vascular damage, atherosclerosis, and thrombosis (32). Also,
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homocysteine impairs cerebrovascular regulation which can effect on the ability of the brain circulation to compensate the blood flre reduction during ischemia. Hcy could compromise vascular regulation by interfering with the action of endothelial vasodilators such as NO and it can also impair compensatory vasodilatory mechanisms and worsen the outcome of cerebral ischemia.
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This hypothesis was proposed after a vascular reactivity to ACh, most likely mediated by release of endothelial NO, which was reduced in hyperhomocysteinemia (34). Another possible
mechanism for the vascular effects of homocysteine was autoxidation of the thiol group, a reaction
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that produces the reactive oxygen species superoxide anion (33).
It has been reported that Hcy reduces nitric oxide (NO) bioavailability by stimulating the formation of reactive oxygen species (34) and increasing matrix metalloproteinase activity,
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which results in alteration of vascular elastin/collagen ratio and compliance reduction of arterial walls (35) and it eventually causes a reduction of vessel radius by thickening arterial wall (16). A significant association between plasma total Homocysteine (tHcy) and increased vascular outcomes was reported previously (16). A relationship between Hcy and cerebrovascular indices has been reported by cross-sectional studies. For example, Lim and colleagues reported a significant relationship between Hcy levels and PI index among patients with ischemic stroke
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(36). In addition, Zhang et al showed that Hcy, at the presence of Cu21, reduces resting cerebral blood flow and attenuates the increases in CBF produced by ACh, hypercapnia, and S-nitroso-Nacetylpenicillamine responses that depend on NO (37). In general, according to different reports,
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a logical relationship between Hcy levels and characteristics of brain vessels either in asymptomatic individuals or patients in different clinical status could be considered. Our study was the first report to investigate the relationship between Hcy and cereberovascular activity
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parameters as well as cognitive status of TBI patients.An inverse association between Hcy level and cognition impairments upon the scales such as MMSE was reported in recent studies (38). The relationship between hHcy and cognitive impairments was related to some conditions such as stroke, Alzheimer's, Parkinson, epilepsy, depression and vascular dementia (11) and, in this study for the first time, we reported a relationship between hHcy and cognitive impairments in TBI patients. In addition, we showed that, there is a significant relationship between Hcy and cerebral hemodynamic abnormality according to PI in TBI patients as the first report in this field. PI is one of the most valuable indicators of early hemodynamic changes obtained by TCD that is
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a good indicator of cerebral vascular resistance (39). Recent studies have reported a direct correlation between PI and prognosis of patients with TBI (40) that was in accordance with our findings. The relationship between Hcy and PI can be linked with other effects of Hcy on the CNS, so that, Hcy by disrupting the function of NO (14) can be involved in the regulation of
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TBI-induced dysregulation of CBF. Of course, this is only a hypothesis and there is no relevant evidence so far in the literature. Furthermore, high level of Hcy has a direct association with up regulation of matrix metalloproteinase’s (MMPs), membrane proteins including tight junctions (TJs) and dysfunction and reduction of endothelial cells forming blood brain barrier (BBB) (34).
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All of these mechanisms can explain the role of Hcy in cereberovascular toxicity (37, 6). We showed a significant correlation between Hcy and cerebral hemodynamic as well as endothelial
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dysfunction makers in TBI patients according to an assessment either in hospital or in six months after discharge. Also, Hcy has a direct association with PI, ICAM-1 and VCAM-1 levels according to in hospital outcome, but a reverse association with CVR in TBI patients. Based on our findings, there is an inverse relationship, between Hcy and markers of cognitive function such as MCA and MMSE six months after discharge in TBI patients. This result was also found for correlation between level of Hcy and CVR. In general, according to our results, we can say
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that Hcy has a direct correlation with outcome of TBI patients. Hcy has also a logical connection parameter with cerebral hemodynamic and endothelial dysfunction markers. The relationship between Hcy and cognitive impairments is another important finding of our study. So far, no specific study has been done in this field and this is the first report on the relationship between
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Hcy and vascular parameters and cognitive performance in TBI patients. Although our study couldn't reveal any direct relation between serum levels of Hcy and patients prognosis but based
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on our findings the levels of Hcy were associated with variables such as GCS, Marshal, CVR and PI that effect on patients' prognosis. Therefore, a possible relation between the levels of Hcy and TBI patients' prognosis could be suggested by our study. This issue requires deep investigations in the field of Hcy in TBI patients. Since the nitrite oxide is one of the markers associated with cereberovascular outoregulation system (42) and its measurement can help to understand the role of Hcy in this system, one of limitations of our study was lack of measurement of NO, and we hope it could be resolved in the future studies. Conflict of interests
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The authors do not have any actual or potential conflict of interests in this study. Acknowledgements
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We thank colleagues in the Faculty of Medicine, Ilam University of Medical Sciences, participants, coordinators and data reviewers who assisted in this study. Funding/Support
Ilam University of Medical Sciences, Ilam, Iran, supported this study. Authors’ contributions
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Hatefi M and Rahmani A and contributed to data analysis, interpretation of the results and drafted the manuscript. Rahmani A participated in the manuscript preparation. Behzadi S participated in radiology
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procedure (TCD, CT-Scan, PI and CVR assessment). Rahmani A, Masoud Dastjerdi MM, Ghahnavieh AA and Madizadeh F participated in data collection. Hatefi M and Hafezi Ahmadi MR contributed to the design of the study and Hafezi Ahmadi MR participated in the laboratory work and manuscript preparation. Hafezi Ahmadi MR and Asadollahi K participated in the manuscript preparation and critical
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review of the paper. All authors have read and approved the article for publication.
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Table 1. Demographic and clinical characteristic of the TBI patients.
29(34.11) 22(25.88) 10(11.76) 6(7.05) 10(11.76) 8(9.41) 5(3-8)
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18(21.17) 67(78.82)
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16(18.82) 13(15.29) 23(27.05) 17(20) 11(12.94) 5(5.88) 10.82± 5.11 6.16± 2.76
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44(51.76) 41(48.23) 54.48±10.34 23.54±13.43
12(14.11) 56(83.58)
18.91±5.67 18.79±4.86 45.58±27.65 863.54±65.75 564.76±33.45 1.03±0.24 3.80±1.49
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Marshall score, n (%) Diffuse I Diffuse II Diffuse III Diffuse IV Evacuated focal mass lesion V Focal mass lesion VI Day in hospitalization Day in ICU Outcome In hospital, n (%) Deceased (GOS 1) Live (GOS 2-5) Outcome In 6 months, n (%) Deceased (GOS 1) Live (GOS 2-5) Cognition Score MCA MMSE Homocysteine (µmol/l) ICAM-1(ng/ml) VCAM-1(ng/ml) PI CVR (%)
TBI , n=85
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Characteristics Sex, n (%) Male Female Age(y) BMI( kg/m2) Injury mechanism Motor Vehicle Accident Motor Cycle Accident Gun Shot Wound Fall Assault Other GCS, median (range)
BMI; Body Mass Index, GCS; Glasgow Coma Scale, ICU; Intensive Care Units, GOS; Glasgow Outcome Scale, MCA; Montreal Cognitive Assessment, MMSE; Mini-Mental State Examination, ICAM; Intercellular Adhesion Molecule-1, VCAM-1; Vascular Cell Adhesion Molecule-1, PI; And Pulsatility Index, CVR; Cereberovascular Reactivity
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Table 2. Association between outcome of Traumatic Brain Injury and Homocysteine, endothelial and cerebral hemodynamic parameters.
58.27±33.48
P
0.045
43.27±26.37
P 0.020
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40.84±26.26
6 Months Outcomes Live Deceased 62.73±12.36
352.80±10.72 361.09±11.11 0.450
352.71±10.64 398.27±13.64 0.000
562.42±24.69 576.45±12.75 0.072
562.63±24.52 603.55±17.31 0.000
1.00±0.28 4.02±1.44 -
0.99±0.28 4.04±1.43 19.77±5.83 19.52±4.81
1.16±0.30 2.73±1.27 -
0.090 0.008 -
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Homocysteine (µmol/l) ICAM1(ng/ml) VCAM1(ng/ml) PI CVR (%) MCA Score MMSE Score
In Hospital Outcomes Live Deceased
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Variables
1.71±0.78 2.15±1.23 13.43±3.33 12.76±5.32
0.000 0.010 0.021 0.026
MCA; Montreal Cognitive Assessment, MMSE; Mini-Mental State Examination, ICAM;
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Intercellular Adhesion Molecule-1, VCAM-1; Vascular Cell Adhesion Molecule-1, PI; And Pulsatility Index, CVR; Cereberovascular Reactivity
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A
B
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Figure 1. Relationship between homocysteine levels and admission Glasgow Coma Scale (A) and the Marshall score (B) in patients with traumatic brain injury 1=Diffuse I, 2=Diffuse II, 3= Diffuse III, 4=Diffuse IV, 5=Evacuated focal mass lesion V 6= Focal mass lesion VI
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A B Figure 2. Multivariate analysis of the effects of homocysteine levels on patients outcome
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A= 6 months outcome; Hcy group (0: <10 µmol/l, 1: 10-30 µmol/l, 2: >30 µmol/l); outcome (0: dead, 1: alive) . B= In hospital outcome; Hcy group (0: <10 µmol/l, 1: 10-30 µmol/l, 2: >30 µmol/l); outcome (0: dead, 1: alive)
40 30 20 10 0
50
*
80 70 60 50
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50
60
40 30
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60
90
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Homocysteine,Mean ±SD(µmol/I)
70
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Homocysteine, Mean±SD(µmol/l)
Homocysteine ,Mean±SD(µmol/I)
70
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20 10
40 30 20 10
0
0
MCA MCA Normal Abnormal
B
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MMSE MMSE Normal Abnormal
C
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Figure 3. Association between Homocysteine (µmol/l) and cognition status according to Montreal Cognitive Assessment (MCA) ( A) , according to Mini-Mental State Examination (MMSE) (B) and pulsatility index (PI) (C) in patients with Traumatic Brain Injury. Data are expressed as Mean ± SD. * =p<0.05.
B
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C
D
Figure 4. Association between Homocysteine and cerebral hemodynamic such as pulsatility index (A) or cereberovascular reactivity (B) and with markers of endothelial dysfunction such as ICAM-1(C) or VCAM-1(D) according to in hospital outcome of patients with traumatic brain injury. ICAM-1; Intercellular Adhesion Molecule-1, VCAM-1; vascular cell adhesion molecule-1.
B
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F
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Figure 5. Association between Homocysteine and markers of cerebral hemodynamic such as pulsatility index (A) and cereberovascular reactivity(B) , and also with markers of endothelial dysfunctions such as ICAM-1(C) and VCAM-1(D) and with cognition score such as MCA score(E) and MMSE score (F) according to six months after discharge from hospital in patients with traumatic brain injury. ICAM-1; Intercellular Adhesion Molecule-1, VCAM-1; vascular cell adhesion molecule-1, MCA; Montreal Cognitive Assessment, MMSE; Mini-Mental State Examination
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Level of Hcy in dead patients after TBI was significantly higher than survived patients in both hospital and six months follow up. Level of ICAM-1, VCAM-1 and PI in deceased patients was higher than survived patients in both hospital and six months follow up in TBI patients. A significant correlation was found between Hcy with cognition impairment according to MCA, A significant correlation was found between Hcy and cerebral
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MMSE in TBI patients.
hemodynamic according to PI in TBI patients. A significant correlation was shown between Hcy
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with ICAM-1 and VCAM-1 in TBI patients.
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Hcy: Homocysteine Hhcy: Hyperhomocysteinemia TBI: Traumatic Brain Injury PI: Pulsatility Indices CVR: Cereberovascular Reactivity MCA: Middle Cerebral Artery TCD: Transcranial Color-Coded Doppler Ultrasonography MCA: Montreal Cognitive Assessment MMSE: Mini-Mental State Examination ICAM-1: Intercellular Adhesion Molecule-1 VCAM-1: Vascular Adhesion Molecule-1 Enos: Endothelial Nitric Oxide CBF: Cerebral Blood Flow GCS: Glasgow Coma Scale BP: Blood Pressure HR: Heart Rate ISS: Injury Severity Score HR: Hormone Replacement Therapy CKD: Chronic Kidney Disease HPLC: High Performance Liquid Chromatography
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List of abbreviations