Predictive value of melatonin and serotonin levels in early recovery period of cerebral ischemic hemispheric stroke

Materials and methods: complex clinical and paraclinical investigation was carried out in 77 patients (the average age of patients was 57.9 ± 0.9 years) on early recovery period of CHIS using of clinical scales (National Institute of Health Stroke Scale, Barthel Index, modified Rankin Scale) on the 10th, 30th, 90th and 180th day of disease, and visualization of cerebral structures by CT scan, and identification of the serum concentration of melatonin and serotonin plasma level on the 10th, 30th day of disease, and also by the calculation of the melatonin/serotonin ratio (MSR) as serum concentration of melatonin divided by serotonin plasma level.


Introduction
Diagnostics and treatment of the patients with cerebral stroke is one of the main problems in modern neurology.Medical and social significance of this problem is caused by the high morbidity, disability and mortality for above mentioned pathology [7].Cerebral stroke is the main reason of disability among surviving patients.Post-stroke disability takes the first place among the reasons of disability concerning adults in the majority countries of the world.80 % of people after brain stroke become disabled, 25 % of people need the nursing care [3].Differentiated determination of optimal structure and number of medical therapies based on the individual prognosis is one of the ways for improvement of functional outcome among the patients with cerebral ischemic stroke [2].
In recent clinical and experimental investigations the role of serotonin [1] and melatonin [4][5][6]8] has been persuasively proved in pathogenesis of acute focal cerebral ischemia.This fact is the basis for suggestion about the possibility to use the humoral markers with aim to predict the outcome of cerebral ischemic stroke.
Its aim was developing criteria for prediction of the outcome during the early recovery period of CIHS on the base of the identification of serum concentration of melatonin and the level of serotonin in the plasma.

Material and methods of investigation
In order to achieve the aim, complex clinical and paraclinical investigation was carried out in 77 patients (55 men and 22 women, the average age was 57.9 ± 0.9 years) on early recovery period of CIHS.The inclusion criteria were: men and women at the age from 33 to 74 years old with the confirmed cerebral hemispheric ischemic stroke according to the data of clinical and computer tomography study; hospitalization during the first 24 hours from the beginning of disease.The patients with the following criterion were excluded from the investigation: acute disorders of cerebral circulation in anamnesis; haemorrhagic transformation of cerebral infarction; combined stroke; ≥2 lesions; somatic pathology on the stage of decompensation; cancer pathology.
The evaluation of the level for neurological deficit was carried out in all the patients according to the National Insti-tute of Health Stroke Scale (NIHSS), the level of functional independence according to Barthel Index (BI) and disability according to modified Rankin Scale (mRS) on the 10 th , 30 th , 90 th and 180 th day of disease.Visualization of cerebral structures was done using the CT scanner Siemens Somatom Spirit (Germany).
Blood samples were collected into 2 vacutainer tubes (one -with clot activator, the next -with EDTA) by venipuncture from median cubital vein on the anterior forearm in the morning (7-7.30AM) on the 10 th and 30 th day from CHIS onset.Melatonin serum level was measured using an immunoassay (IBL, Germany, Cat.No. RE54021) at Scientific Medical Laboratory Center (head -prof.A. Abramov) of ZSMU.Serotonin plasma level was measured using a fluorescent spectrophotometry (λex = 300 nm, λem = 540 nm) at Laboratory Diagnosis and General Pathology Department (head -prof.A.Trailin) of the Zaporizhzhia Medical Academy of Postgraduate Education.Melatonin/Serotonin ratio (MSR), dynamic coefficients of melatonin (ΔME), serotonin (ΔSE) and MSR (ΔMSR) changes from the 10 th day to the 30 th day as compared to the 10 th day were calculated.
Statistical data processing was done using the Statistica 6.0 (StatSoft Inc., USA, serial number AXXR712D-833214FAN5) application.Descriptive statistics are presented as median and interquartile range -Ме [Q1; Q3].The criteria of Mann-Whitney and Kruskal-Wallis were used for the estimation of intergroup differences, binary logistic regression and ROC-analysis were used for development of the predicting criteria.

Results and their discussion
On the 10 th and 30 th day of disease essential differences of serum concentration of melatonin, plasma concentration of serotonin and their correlation among the patients with CIHS depending on the age and sex were not revealed.
Distribution of patients with CIHS depending on the mRS index on the 90 th and 180 th day of disease is revealed in the Table 1.
As the primary endpoints, the cases of moderate and profound disability on the 90 th and 180 th day of the disease according to the indexes of mRS ≥ 3 points were registered respectively in 14 (18.2 %) and 12 (15.6 %) patients.Using the comparative ROC-analysis we defined that the most informative parameters for prediction of moderate and severe disability for mRS ≥ 3 points on the 90 th day of CIHS are the following (in decreasing order of predictive value): the level of MSR on the 30 th day (AUC = 0.81, p < 0.05), the serotonin plasma level on the 30 th day (AUC = 0.69, p < 0.05), dynamics of MSR level on the 30 th day (AUC = 0.67 p < 0.05), the serotonin plasma level on the 30 th day (AUC = 0.65, p < 0.05) and dynamics of serum concentration of melatonin on the 30 th day (AUC = 0.64, p < 0.05) (Table 2).

Оригінальні дослідження
In accordance with the date from the table 2, the most informative parameters for prediction of moderate and severe disability for mRS ≥ 3 points on the 180 th day are also MSR level on the 30 th day(AUC = 0.78, p < 0.05), the serotonin plasma level on the 30 th day (AUC = 0.74, p < 0.05), dynamics of serum concentration of melatonin on the 30 th day (AUC = 0.67, p < 0.05), dynamics of the serotonin plasma level on the 30 th day (AUC = 0.67, p < 0.05), dynamics of MSR on the 30 th day (AUC = 0.66, p < 0.05) and the serum concentration of melatonin on the 30 th day (AUC = 0.66, p < 0.05).
On the basis of ROC-analysis the definitions of above mentioned indexes for prediction of moderate and severe disability for mRS ≥ 3 points on the 90 th day (Table 3) and on the 180 th day of CIHS (Table 4) with the optimal correlation of sensitivity and specificity were determined.
It was defined that prognostic criterion of moderate and severe disability for mRS ≥ 3 points on the 180 th day of CIHS were: MSR level on the 30 th day ˃212.0,MSR dynamics on the 10 th -30 th day ˃1.019, the serum concentration of melatonin on the 30 th day ≤29.1 pg/ml and dynamics of serum concentration of melatonin on the 10 th -30 th day ≤0.382,whereas the level of serotonin in blood plasma on the 30 th day is ˃0.15 mcmol/l and its dynamics on the 10 th -30 th day ≥-0.318 were associated with the mRS ≤2 points on the 180 th day of disease.
So, decreasing of serum concentration of melatonin on the 30 th day or its increasing no more than 38.2 % from the level on the 10 th day of disease are prognostically unfavorable for the functional outcome during early recovery period of CIHS.It allows considering mentioned options of melatonin dynamics as the markers of insufficiency in the system of endogenous antioxidant protection.At the same time it is the base for more intensive neurometabolic and antioxidant therapy among the patient of such a group.
Moreover, decreasing of serotonin level in blood plasma on the 30 th day more than 31.8 from its level on the 10 th day of the disease is also prognostically unfavorable factor.The availability of correlation interaction between plasma serotonin concentration and its level in brain was proved earlier by T. Audhya with co-authors [9].Received data is also coordinated with the results of I. V. Shilonosova's investigation (2012), where inverse correlation between the content of humoral serotonin on the 16 th -24 th day of CIHS and the level of neurological deficit was defined [1].Taking into consideration the ability of serotonin to play the role of angiogenesis inductor [4] and revealed by us interconnection between its concentration in blood and forecast of CIHS treatment, we can affirm about the pathogenetic significance of serotonin in the realization of sanogenesis mechanisms during early recovery period of CIHS.
Among the studied indexes the highest information value for prognosis of functional outcome of early recovery period of CIHS as for the results of this research was set for the ratio of melatonin and serotonin on the 30 th day of disease (AUC = 0.81 against 0.45 и 0.69 for melatonin and serotonin respectively).It defines the practical usage of mentioned criterion as surrogate marker of objectification of interconnection of melatonin and its metabolic precursor -serotonin among the patients with CIHS as

Conclusions
Conducted investigation allows us to draw out the following conclusions: 1.The level of MSR ˃212.0 on the 30 th day of CIHS is the predictor of moderate and profound disability for modified Rankin's scale ≥3 points on the 90 th (AUC = 0.81, p ˂ 0.05; sensitivity = 100.0%, specificity = 75.0%) and on the 180 th day of disease (AUC = 0.78, p ˂ 0.05; sensitivity = 100.0%, specificity = 73.3%).
2. The serotonin plasma level ≤0.15 mcmol/l on the 30 th day of CIHS is the predictor of moderate and profound disability for modified Rankin's scale ≥3 points on the 180 th day of disease (AUC = 0.74, p ˂ 0.05; sensitivity = 60.0 %, specificity = 86.7 %).
Perspective for further scientific investigations is the development of differential approach for arranging treatment and rehabilitation in patients with CIHS, taking into account the individual prognosis of outcome on the early recovery period of disease, based on the identification of the serum concentration of melatonin and the serotonin plasma level.

Table 1 .
Distribution of patients with CIHS depending on the mRS index on the 90 th and 180 th day of disease

Table 2 .
Comparative analysis of informational content as for melatonin and serotonin levels, MSR on the 10 th and 30 th day of CIHS for prediction of moderate and severe disability for mRS ≥ 3 points on the 90 th and 180 th day of disease on the basis of calculations of Area Under the Curve index (AUC) mRS: modified Rankin Scale score; *: significance of differences with the value of AUC = 0.5; p < 0.05.

Table 3 .
Criteria for prediction of moderate and severe disability for mRS ≥ 3 points on the 90 th day of CIHS

Table 4 .
Criteria for prediction of moderate and severe disability for mRS ≥ 3 points on the 180 th day of CIHS