Analysis of rat blood plasma upon acute epileptic seizures by infrared spectroscopy with chemometrics
Introduction
Epilepsy is a chronic neurological disorder characterized by repetitive and unprovoked seizures which result in various symptoms [1,2]. As affecting approximately 70 million people worldwide, it is defined as one of the major global health issues by World Health Organization since there is no complete treatment due to the limited knowledge on its complicated and multifactorial mechanisms [3]. Despite current anti-epileptogenic strategies, epileptic seizures can be controlled only in about 70% of cases [2,4]. The goal of management and treatment of this disorder is to achieve seizure free-status and to determine who is going to develop epilepsy before epileptic seizure manifest, which totally depends on an early and accurate diagnosis and intervention [2,5]. To achieve this, clinical and experimental research have been historically continued to identify the potential biomarkers that are signs of the factors involved in generations and propagations of seizures [[5], [6], [7]]. Thus, the elucidation of such biomarkers provides insights into underlying fundamental mechanisms of epileptogenesis and ictogenesis, which could serve as targets for anti-epileptogenic strategies [5]. Furthermore, the proper validation of epileptic biomarkers give additional opportunities such as measurement of progression after establishment of the condition, screening of potential anti-epileptogenic drugs, and determination of pharmaco-resistance [[5], [6], [7]].
Besides brain tissue, blood also enables to give specific information about metabolic response to provocation of epileptic seizures. Relatively, the processes involved in epileptogenesis may reveal altered blood profile as shown in different reports [5,[8], [9], [10], [11], [12]]. For example, epilepsy induced-changes such as different amino acid concentrations [12,13], an increment in specific proteins [14], and high level of some cytokines [15,16] were observed in animal and human studies. In addition, some variations in blood lipids were shown to relate to the occurrence of epileptic seizures [[17], [18], [19], [20]]. All these alterations in blood profile may offer predictive results for epileptic condition, as suggested in literature [8,10,11].
Blood monitoring has some advantages since it can be repeated routinely as well as it provides convenient, cost-saving and minimally invasive method for follow-up screening of patients suffering from epilepsy [21,22]. For that reason, the effect of epileptic seizures on blood constituents has received increased attention [5,[8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]]. However, routinely applied blood analyzers can only measure few biochemical parameters. Considering that generation and consequences of epileptic seizures are dependent on multifactorial mechanisms, the methods giving general information are required to present an overview about the processes in blood. Fourier transform infrared (FT-IR) spectroscopy offers suitable approaches for such analysis. This technique has benefits over analytical methods as being rapid, easy to use, sensitive, minimal sample preparation requirement, automated algorithms usage, and operating without external markers [[23], [24], [25], [26], [27], [28], [29], [30], [31]]. For this reason, it has been offered as a complementary method to analyse blood components after a variety of diseases and pathological conditions [23,24,[27], [28], [29], [30], [31], [32], [33], [34], [35]]. In particular, by performing multivariate statistics the simultaneous implication of different spectral data is facilitated, and thus the accuracy and predictive capability of the analysis is improved [25,26,[28], [29], [30],34].
To the best of our knowledge, FT-IR spectroscopy with multivariate analysis has not been performed on blood sampling in any epileptic condition. In the present study, we aimed to analyse blood plasma after acute epileptic seizures using a rat model experienced pentyleneterazol (PTZ)-induced generalized tonic-clonic seizures (GTCS). In the scope of the study, the second derivate spectra were evaluated and classified by principle component analysis (PCA) and hierarchical cluster analysis (HCA). Since there are reports stating epileptic conditions may cause to alter peripheral blood in terms of lipids and proteins, we specifically focused on 3000-2800 cm−1 and 1730-1480 cm-1 spectral ranges.
Section snippets
Animal experiment and sample preparation
Adult male Wistar rats were used in the experiments. All applicable international, national, and/or institutional guidelines (EU Directive 2010/63/EU) for the care and use of animals were followed and approved by the Ethics Committee (KOU44543) of Kocaeli University, Turkey. The animals were allowed to access to food and water, and were kept under 12 h light/dark cycle with humidity of 10 − 50%. They were separated into (1) control group (n = 7) and GTCS-group (n = 7). Control group were
Results and discussion
Evidences show that altered peripheral blood profile is one of consequences of epileptic seizures [5,[8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]]. When combined with appropriate chemometric analysis FT-IR spectroscopy offers novel and suitable approach for such biomolecular changes as also shown for other pathological conditions [[23], [24], [25], [26], [27], [28]]. In this study, we utilized FT-IR spectroscopy together with HCA and PCA on analysis of blood plasma
Conclusions
By taking advantages of FT-IR spectroscopy, we previously obtained significant molecular alterations in brain tissues and membranes after acute epileptic seizures, and further, successfully discriminated seizure group from control by application of chemometrics [25,26]. Similarly, considering altered peripheral blood components during epileptic conditions, we analyzed rat blood plasma after acute GTCS by FT-IR spectroscopy together with HCA and PCA.
Our findings showed that, unsuccessful
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
CRediT authorship contribution statement
Sevgi Türker-Kaya: Investigation, Writing - original draft, Writing - review & editing, Supervision. Gül İlbay: Resources, Investigation, Writing - original draft.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
We would like to thank Aygül Kına for FT-IR measurements of plasma samples.
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