Biomarkers of traumatic brain injury

Abstract

To provide a method of diagnosing and / or monitoring traumatic brain injury (TBI) in a subject.SOLUTION: A method comprises determining a level of at least one miRNA in a fluid sample from the subject. The miRNA may be selected from miR-425-5p, miR-502, miR-21 and miR-335. The method may involve determining whether a subject is suffering from mild-TBI or moderate to severe TBI. Also, provided are a sensor element, a detection system and a kit for diagnosing and / or monitoring TBI, and a method of determining an appropriate treatment for a subject with suspected TBI.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to compositions, kits, systems and methods for diagnosing and / or monitoring traumatic brain injury (TBI). More particularly, the present invention relates to the diagnosis and monitoring of TBI using miRNA biomarkers.

Background Art

[0002] Traumatic brain injury (TBI) is a leading cause of death and physical disability in people under 45 years old in Western countries. Its medical cost burden and social costs are expected to continue to rise, and the World Health Organization estimates that by 2020, TBI will be the third leading cause of physical disability worldwide.

[0003] Despite numerous studies, no reliable biomarkers have been found to assess the severity of TBI and predict recovery. This is particularly true for mild traumatic brain injury (mTBI), which is still difficult to evaluate in clinical practice. TBI patients are initially evaluated by the Glasgow Coma Score (GCS) and neuroimaging techniques, but these require expensive equipment, and current diagnostic tools lack the ability to precisely define and quantify the actual severity of brain injury. That is, severe TBI is easily detected by them, but mTBI, which accounts for the majority (75-90%) of cases, is not easily detected.

[0004] An accurate diagnosis of mTBI is particularly important in patients such as athletes, military personnel and children, who are at high risk of a dramatic form of brain injury known as second impact syndrome (SIS), which can cause severe damage and even death due to repeated mTBI and the synergistic effects of repeated TBI. That is, early diagnosis of TBI and assessment of its severity are very important for the well-being of patients and ultimately to save the lives of patients.

[0005] The need for TBI biomarkers has gained significant momentum due to the increasing attention in the mass media on sports concussion. In recent years, numerous studies have focused on biomarkers that can support clinical decision-making in the sidelines or in sports clinics. However, the protein biomarkers reported in the literature lack either specificity or sensitivity, or are undetectable for some time after injury. This may be due to the fact that after concussion, a form of TBI, very little brain-derived compounds are released and the blood-brain barrier remains mostly closed.

[0006] MicroRNA (miRNA) is a large class of highly conserved non-coding RNA molecules approximately 22 nucleotides in length that cause mRNA degradation, translational repression, or both, through pairing with partially complementary sites in the 3'UTR of target genes. The human genome encodes over 2000 miRNAs, which may target approximately 60% of all genes. However, despite their abundance, their biomolecular functions and involvement in etiology are not fully understood. miRNAs play a central role in numerous biological processes including the cell cycle, cell metabolism, apoptosis and immune response, and are gaining increasing attention in clinical research as potential biomarkers for the detection, identification and classification of other disease states including cancer and neurodegenerative diseases.

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present invention has been devised in view of these matters.

[0008] The present invention provides a method for diagnosing or monitoring traumatic brain injury (TBI) in a subject.

Means for Solving the Problems

[0009] According to a first aspect of the present invention, there is provided a method for diagnosing and / or monitoring traumatic brain injury (TBI) in a subject, comprising the step of detecting the presence of and / or determining the level of at least one miRNA in a sample derived from the subject.

[0010] At least one miRNA (also referred to herein as "miR") is selected from the group consisting of miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-335, miR-126 * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 * , miR-671-3p, hsa-let-7c-5p, hsa-let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p, miR-221-3p, miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p, miR-424-5p, miR-30a-5p; miR-107; miR-135b-5p; miR-199b-5p; miR-324-5p; miR-652-3p; miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p and miR-629. These miRNAs may be referred to herein as the miRNA of interest or target miRNA.

[0011] In some embodiments, at least one miRNA is selected from the group consisting of miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-335, hsa-miR-126 * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 * , and miR-671-3p. These microRNAs have been found to be biomarkers expressed in all TBI patients (mild or severe).

[0012] In some embodiments, at least one miRNA is selected from the group consisting of miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g and miR-335.

[0013] In some embodiments, at least one miRNA is selected from the group consisting of let-7c-5p, let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p, miR-221-3p, miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p, and miR-424-5p; miR-30a-5p; miR-107; miR-135b-5p; miR-199b-5p; miR-324-5p; miR-652-3p.

[0014] In some embodiments, at least one miRNA is selected from the group consisting of miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-194, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p, and miR-629.

[0015] To avoid ambiguity, "at least one miRNA is selected from a group of miRNAs" means, as used herein, that for any diagnostic, prognostic, or therapeutic purpose for which the method in question is performed, any one of the listed miRNAs or any plurality of the listed miRNAs (e.g., 2, 3, 4, or more of the listed miRNAs) can be used. As a result, one or more of the listed miRNAs may be explicitly excluded. For example, if at least one miRNA is selected from the group consisting of miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, and miR-335, the method can include detecting and / or evaluating the level of any combination of miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, and miR-let-7g, excluding miR-335.

[0016] According to one aspect of the present invention, there is provided a method for diagnosing and / or monitoring traumatic brain injury (TBI) in a subject, comprising determining the level of at least one miRNA in a sample derived from the subject, wherein the miRNA is selected from the group consisting of miR-425-5p; miR-502; miR-21; and miR-335.

Brief Description of the Drawings

[0017]

Figure 1

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Figure 3B

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Figure 4B

[0018] Traumatic brain injury occurs when an external force traumatically injures the brain. For example, there are various systems for classifying TBI based on severity, type of injury, and prognosis. The most commonly used system for classifying TBI is the Glasgow Coma Scale (GCS), which grades a person's level of consciousness on a scale of 3 - 15 based on verbal response, motor response, and eye-opening response to stimuli. Generally, a TBI with a GCS score of 13 or higher is defined as mild, 9 - 12 as moderate, and 8 or lower as severe. Another system, the Mayo classification system, has three main classifications, including definite moderate to severe TBI, presumed mild TBI, and possible TBI. Multiple criteria are used in each diagnosis, including criteria such as loss of consciousness, post-traumatic amnesia, skull fracture, and signs of neuro-radiological abnormalities such as subdural hematoma, cerebral contusion, and hemorrhagic contusion. Classification of TBI using the GCS or Mayo system will be known to those skilled in the art.

[0019] As used herein, references to "mild," "moderate," and "severe" TBI are made according to the GCS. References herein to "moderate to severe" TBI include both moderate and severe TBI according to the GCS.

[0020] The diagnosis and / or monitoring of TBI using the biomarkers of the present invention is expected to assist clinical decision-making and treatment regimens in various contexts, including but not limited to: as part of the paramedical initial assessment to determine whether to transfer a patient to a facility with neurosurgical expertise, a large trauma center or a local trauma unit; in the hospital emergency department to determine appropriate treatment, including the need for a CT brain scan; at the side of the playing field to assist in the decision to remove a player from competition and evaluate the need to transport the player to the hospital; in the sports outpatient clinic to confirm a concussion event and enable decisions regarding return to play; and in combat situations to determine the need to dispatch a rescue team and evacuate victims. That is, the subjects to whom the present invention provides specific benefits include victims of accidents, sports players, and military personnel.

[0021] In any case, particularly perhaps when the subject has a relatively high risk of TBI (e.g., when the subject is a professional sports player or enlisted in the military), a sample can be obtained from the subject at any known or recent pre-injury time point (e.g., near the start of a sports career or before military deployment), and any miRNA of interest can be evaluated at or after the time point when the subject may have suffered TBI. Such a sample can thereby provide an internal reference standard.

[0022] In some embodiments, the subject is human.

[0023] TBI can be mild TBI (mTBI), moderate TBI or severe TBI (sTBI). In some embodiments, TBI is moderate to severe TBI (m-sTBI).

[0024] The level of miRNA or each miRNA in a sample can be determined quantitatively or semi - quantitatively. By "quantitatively", it will be understood that the absolute amount or concentration of miRNA or each miRNA in the sample is determined. The absolute amount of miRNA or each miRNA in the sample can subsequently be compared to a predetermined threshold (e.g., published literature values regarding the predicted normal level), the known level of the same or reference miRNA in a control sample taken from a healthy subject, or the amount of reference miRNA in a sample taken from the subject. In some embodiments, a subject is diagnosed with TBI if the level of miRNA is less than a predetermined threshold or is decreased compared to a reference sample or control sample. In other embodiments, a subject is diagnosed with TBI if the level of miRNA is increased compared to a predetermined threshold.

[0025] By "semi - quantitatively", it will be understood that the level of the miRNA or each miRNA of interest is measured in comparison to a reference.

[0026] The reference can be a non - variable miRNA, i.e., a miRNA having an expression level that remains substantially unchanged between healthy subjects and subjects with TBI. A subject can be diagnosed as suffering from TBI if the level of the miRNA or each miRNA of interest is increased or decreased compared to that of the non - variable miRNA. Suitable non - variable miRNAs include miR - 331, miR - 223 * , miR - 23a - 3p and miR148b - 3p. miR - 23a - 3p and miR148b - 3p are invariant only in saliva.

[0027] In some embodiments, the level of miRNA or each miRNA in a sample obtained from a subject can be about 0.01 - fold to about 100 - fold, about 0.05 - fold to about 50 - fold, about 0.1 - fold to about 10 - fold, about 0.5 - fold to about 5 - fold, about 1.0 - fold to about 3 - fold, or about 1.5 - fold to about 2.0 - fold lower or higher than the level in a control sample, a reference level, or a published value.

[0028] When a device or method is used to generate a value, the inventors may condition the value with the term "about" in order to obtain the described value and any variability of that value specific to the device or method used. When a value or range of values is specifically disclosed, "about" can mean ±10% of the recited value or range. For example, about 10 minutes can mean 9 to 11 minutes.

[0029] The miRNA of interest or the level of each miRNA can be determined using methods known to those of ordinary skill in the art. In some embodiments, the step of determining the miRNA of interest or the level of each miRNA includes the step of amplifying the miRNA. In some embodiments, total miRNA can first be isolated from a sample using standard techniques, e.g., using the miRNeasy mini kit (Qiagen). The amount of miRNA of interest can then be determined. In some embodiments, the level of the miRNA of interest or each miRNA in a sample is determined using PCR (polymerase chain reaction). For example, quantitative PCR can be used for the quantitative determination of the level of the miRNA of interest or each miRNA. PCR can also be used for semi - quantitative determination by comparing the level of the miRNA of interest or each miRNA in a sample to that of a reference (e.g., an invariant miRNA).

[0030] Techniques suitable for miRNA detection and / or quantification will be known to those of ordinary skill in the art and include, for example, qPCR, miRNA assays, next - generation sequencing (NGS), and multi - miRNA profiling assays.

[0031] In some embodiments, the level of the miRNA of interest or each miRNA is determined using in - situ hybridization, e.g., using a probe specific for the miRNA (e.g., a labeled probe).

[0032] The miRNA levels can be determined in samples obtained from a subject immediately after injury (i.e., less than 1 hour after injury), and / or at one or more time points several hours or days after injury. That is, changes in miRNA levels can be detected over time to enable monitoring of TBI. If the miRNA levels change over time, the methods for monitoring TBI described herein can be extended to include steps of maintaining or adjusting the treatment regimen of the subject accordingly.

[0033] Depending on the specific miRNA and type of TBI, the levels of miRNA in a subject may change significantly over time. In some embodiments, therefore, it may be advantageous to measure the miRNA relatively soon after injury to enable an accurate diagnosis. In some embodiments, the miRNA levels are determined in samples obtained from a subject within 72 hours or less, 48 hours or less, 36 hours or less, 24 hours or less, 12 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, or 1 hour or less after injury.

[0034] The levels of some miRNAs are substantially stable over time, thus enabling diagnosis even several hours, days, or weeks after injury. In some embodiments, the miRNA levels are determined in samples obtained from a subject up to 20, 18, 15, 12, 10, 8, 5, or 2 days after injury.

[0035] In some embodiments, the miRNA levels are determined in samples obtained from a subject immediately after injury (e.g., T = 0 h), 4 - 12 hours after injury, 48 - 72 hours after injury, or 15 days after injury.

[0036] In some embodiments, the TBI is a mild TBI (mTBI) or a moderate-to-severe TBI (m-sTBI), and at least one miRNA is selected from the group consisting of miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-335, hsa-miR-126 * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 * , and miR-671-3p

[0037] In some embodiments, the TBI is a mild TBI (mTBI), and the miRNA is selected from the group consisting of miR-425-5p and miR-502. A subject can be diagnosed with mTBI if the level of miR-425-5p and / or miR-502 is determined to be below a predetermined threshold or is decreased compared to a reference.

[0038] In some embodiments, the levels of miR-425-5p and / or miR-502 that are below a predetermined threshold or are decreased compared to a reference are those determined in a sample obtained within less than 48 hours after the injury for diagnosing mTBI.

[0039] In some embodiments, the TBI is a moderate-to-severe TBI (m-sTBI), and the miRNA is selected from the group consisting of miR-21 and miR-335. A subject can be diagnosed with moderate-to-severe TBI if the level of miR-21 and / or miR-335 is determined to exceed a predetermined threshold or is increased compared to a reference.

[0040] In some embodiments, levels of miR-21 and / or miR-335 that exceed a predetermined threshold or are increased compared to a reference, when measured in a sample obtained up to 15 days after injury, diagnose moderate to severe TBI.

[0041] In some embodiments, the TBI is moderate to severe TBI (m-sTBI), and at least one miRNA is selected from the group consisting of miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-194, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p, and miR-629.

[0042] If the levels of miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-618, miR-95, miR-130a, miR-152, miR-194, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, and / or miR-99a are determined to exceed a predetermined threshold or are increased compared to a reference, the subject can be diagnosed as having m-sTBI.

[0043] If the levels of miR-192, miR-429, miR-520D-3p, and / or miR-629 are determined to be below a predetermined threshold or are decreased compared to a reference, the subject can be diagnosed as having m-sTBI.

[0044] The miRNAs can be used individually to diagnose TBI. For example, in sports concussion, miR-502 or miR-425-5p could be used to confirm the occurrence of traumatic brain injury.

[0045] That is, a further aspect of the present invention is a method for determining the severity of TBI, and the steps of this method can be repeated to monitor a subject over time. A positive result for a single miRNA (e.g., the level of a single miRNA is determined to be above a predetermined threshold / below a predetermined threshold, or increased / decreased compared to a reference) will be understood to be sufficient to determine the severity of TBI. For example, if the level of miR-425-5p is below a predetermined threshold or decreased compared to a reference, the severity of TBI is determined to be mild (mTBI). However, it may be convenient to combine different miRNAs (e.g., in a test panel) to facilitate the assessment of TBI severity.

[0046] In some embodiments, the method includes determining the levels of a plurality (e.g., two or more) miRNAs in a sample. In some embodiments, the two or more miRNAs are selected from the group consisting of miR-425-5p; miR-502; miR-21; and miR-335.

[0047] In some embodiments, the method is the following miRNAs: (i) a first miRNA selected from miR-425-5p and miR-502; and (ii) a second miRNA selected from miR-21 and miR-335 and includes determining the levels thereof.

[0048] If it is determined that the level of miR-425-5p or miR-502 is below a predetermined threshold or decreased compared to a reference, or if it is determined that the level of miR-21 or miR-335 is above a predetermined threshold or increased compared to a reference, the subject can be diagnosed as having TBI.

[0049] In some embodiments, the TBI is a mild TBI (mTBI), and at least one miRNA is selected from the group consisting of let-7c-5p, let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p, miR-221-3p, miRmmiR-29a-3p, miR-29c-3p, miR-424-5p, miR-30a-5p, miR-107, miR-135b-5p, miR-199b-5p, miR-324-5p, and miR-652-3p, or combinations thereof. When the fold change in the level of microRNA compared to the reference is at least 0.5, at least 1.0, at least 1.5, at least 2.0, at least 2.5, at least 3.0, at least 3.5 or at least 4.0, the subject can be diagnosed as having mTBI. In some embodiments, when the level of microRNA is increased compared to the reference, the subject is diagnosed as having mTBI.

[0050] The sequences and accession numbers for the miRNAs described herein are provided in Table 1:

Table 1

[0051] Briefly, the sample can be any suitable bodily fluid or tissue sample obtained from a subject. For example, biological samples can include at least one of the group consisting of the following samples: urine, saliva, whole blood, plasma, serum, sputum, semen, feces, nasal swabs, tears, vaginal swabs, rectal swabs, cervical smears, tissue biopsies, and urethral swabs. In some embodiments, the sample is a bodily fluid sample. Preferably, the sample is one that can be easily obtained from an individual, such as urine, saliva, blood, and sputum. In some embodiments, the sample includes saliva, blood, plasma, or serum. It will be understood that in some embodiments, the process of obtaining the sample does not form part of the invention described herein.

[0052] In some embodiments, the sample comprises or consists of serum. Serum not only has practical advantages but also does not contain anticoagulant components such as heparin, which are potential inhibitors of the PCR reaction. Serum may also be less susceptible to the effects of hemolysis compared to plasma.

[0053] In some embodiments, the sample is saliva. Saliva can be easily obtained from a patient without the use of expert training or medical equipment (e.g., beside a playing field or in the wild).

[0054] The miRNAs found in saliva that indicate mTBI are: hsa-let-7ca-5p, hsa-let-7i-5p, hsa-miR-1421-3p, hsa-miR-148a-3p, hsa-miR-15b-5p, hsa-miR-16-5p, hsa-miR-181a-5p, hsa-miR-20a-5p, hsa-miR-20b-5p, hsa-miR-221-3p, hsa-miR-24-3p, hsa-miR-27b-3p, hsa-miR-29a-3p, hsa-miR-29c-3p, hsa-miR-340-5p, hsa-miR-424-5p; miR-30a-5p; miR-107; miR-135b-5p; miR-199b, and are selected from the group consisting of -5p; miR-324-5p; and miR-652-3p.

[0055] The levels of miRNA can be used to track the subject's recovery from the injury. That is, the present invention encompasses monitoring the subject's recovery from TBI as an alternative to, or in addition to, an initial diagnosis.

[0056] In some embodiments, the method includes the step of monitoring TBI, and the level of at least one miRNA is determined in a sample obtained from the subject at least 2 days, at least 3 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days after the injury. In some embodiments, the level of at least one miRNA is determined in a sample obtained from the subject 15 days after the injury. In some embodiments, the level of at least one miRNA is determined in at least two samples obtained at different time intervals after the injury, thus enabling monitoring of the recovery. For example, the miRNA levels could be determined 7 days and 14 days after the injury, or 5 days, 10 days, and 15 days after the injury. The return of the miRNA levels to normal values may indicate the subject's recovery from TBI.

[0057] In some embodiments, a subject is determined to have recovered from mTBI if the levels of miR-425-5p and / or miR-502 are no longer below a predetermined threshold or are no longer decreased compared to a reference.

[0058] In some embodiments, a subject is determined to have recovered from moderate to severe TBI if the levels of miR-21 and / or miR-335 are no longer above a predetermined threshold or are no longer increased compared to a reference.

[0059] Diagnosis of a subject suffering from TBI, particularly mild TBI or moderate to severe TBI, may facilitate the determination of appropriate treatment. That is, the present invention provides a test that enables medical professionals such as surgeons, clinicians, paramedicals, and even non-medical persons (e.g., teachers, sports coaches, military personnel) to decide on appropriate actions for a subject suspected of having TBI. A subject determined to have TBI can thus receive the most appropriate treatment as a result of the diagnosis made. That is, the method of the present invention can further include the step of causing a subject diagnosed with TBI to receive appropriate therapy.

[0060] A subject diagnosed with TBI can be further evaluated, for example, by a CT scan. In some embodiments, the subject is hospitalized. In some embodiments, if moderate to severe TBI can be ruled out, the subject may not need to be hospitalized for evaluation. A subject diagnosed with moderate to severe TBI can be hospitalized in a hospital or a specialized center with expertise in traumatic brain injury.

[0061] A subject diagnosed with TBI (particularly mTBI) outside a hospital environment, e.g., during a sports event, combat, or competition, can be promptly excluded from the competition or combat. Subsequently, the subject can be started on a gradual return to the competition or combat.

[0062] In a further aspect, the following steps: determining the level of at least one miRNA in a sample from a subject; and determining whether it is appropriate to administer a therapy to reduce TBI based on the level of the at least one miRNA are provided for determining whether it is appropriate to administer a therapy to a subject to reduce TBI.

[0063] It will be understood that the step of administering a therapy to a subject does not form part of the claimed method unless specifically recited.

[0064] In some embodiments, the method can further comprise the step of administering a suitable treatment to the subject. In some embodiments, the treatment can include a therapy to reduce TBI. Accordingly, the present invention features a method of diagnosing and treating TBI in a subject, comprising the steps of: (a) obtaining a sample (e.g., a sample of blood, plasma, serum, urine, or saliva) from the subject; (b) detecting one or more miRNAs (selected from those described herein); diagnosing the patient as having TBI if the level of the miRNA is different from a reference standard (as described herein); and administering a treatment for TBI.

[0065] In a further aspect, the present invention provides a method for determining an appropriate treatment for a subject suspected of suffering from TBI, comprising determining whether the subject has TBI by determining the level of at least one miRNA in a sample from the subject.

[0066] If the subject is identified as having TBI, appropriate treatments can include one or more of the following: further tests (e.g., language tests, cognitive tests, motor tests and / or visual tests), further evaluation of the subject by CT and / or MRI scans; excluding the subject from activities (e.g., the activity during which the TBI occurred); admitting the subject to a hospital or a specialized outpatient clinic; surgery; and administering to the subject therapies to reduce the TBI.

[0067] Therapies to reduce the TBI can include neuroprotective agents, drugs for treating brain swelling such as mannitol and hypertonic saline, and / or other neuroprotective means such as avoidance of hypertensive resuscitation and use of sedatives.

[0068] In some embodiments, the subject can subsequently be monitored, e.g., in a hospital or clinical setting, to track its recovery.

[0069] According to a further aspect of the invention, there is provided a method for detecting and / or determining the level of a target miRNA in a subject, comprising the steps of: (a) obtaining a sample from the subject; and (b) detecting and / or determining the level of the target miRNA in the sample by contacting the sample with a probe specific for the target miRNA.

[0070] The sample can be any suitable body fluid or tissue sample obtained from the subject as defined above. In some embodiments, the sample is blood, serum, plasma, urine or saliva.

[0071] In some embodiments, the method can include the step of determining the levels of two or more target miRNAs in the sample.

[0072] According to a further aspect of the invention, there is provided a therapy for reducing TBI for use in a method of treating a subject in need thereof, wherein the subject has been identified as having TBI by determining the level of at least one miRNA in a sample from the subject.

[0073] The step of determining the level of the target miRNA can include contacting a sample with a substrate functionalized with a probe, such as a chip comprising the probe. The substrate or chip can advantageously include a plurality of probes each specific for a different target miRNA.

[0074] The subject can be suffering from an injury, particularly a head injury. The subject may be suspected of having TBI. In some embodiments, the sample is obtained within 72 hours or less, 48 hours or less, 36 hours or less, 24 hours or less, 12 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, or 1 hour or less after the injury.

[0075] In some embodiments, the method further includes the step of treating the subject. Treatments include one or more of the following: further evaluating the subject, for example, by additional tests (such as speech tests, cognitive tests, motor tests, and / or vision tests), CT and / or MRI scans; excluding the subject from activities (such as the activity during which the TBI occurred); admitting the subject to a hospital or a specialized outpatient clinic; and administering to the subject a therapy for reducing TBI. In some embodiments, the treatment includes administering an effective amount of a neuroprotective agent.

[0076] That is, in a further aspect, the invention includes the steps of: determining the level of at least one miRNA in a sample from the subject; and when the level of the at least one miRNA indicates mTBI, administering an appropriate treatment for mTBI; or When the level of said at least one miRNA indicates m-sTBI, steps of administering appropriate treatment to m-sTBI A method of treating TBI is provided, including

[0077] It will be understood by those skilled in the art that different treatment pathways can be used for mTBI and m-sTBI.

[0078] Appropriate treatments for mTBI include the following treatments: excluding the subject from activities; treatment at the scene or within the area; further evaluating the subject in a hospital without overnight hospitalization (typically, mTBI patients are discharged quickly after receiving a head injury notification); or hospitalization in a hospital for an observation period (typically 1-2 days). The subject can be further evaluated using tests (e.g., language tests, cognitive tests, motor tests, and / or visual tests). CT scans are generally required only when specific signs are present, such as suspected skull fracture, post-traumatic seizure, focal neurological deficit, repeated vomiting, and a GCS score of less than 13 (less than 14 for children, or less than 15 for infants less than 1 year old) in the initial evaluation, in accordance with NICE guidelines.

[0079] Appropriate treatments for m-sTBI include the following treatments: MRI or CT scan (especially within 1 hour of injury); hospitalization in a hospital (which may include admission to an intensive care unit and / or transfer to a large trauma center with a specialized outpatient or neurosurgical facility); neurological monitoring; surgery; administration of neuroprotective drugs (e.g., drugs for treating brain swelling such as mannitol and hypertonic saline), and / or other neuroprotective measures such as avoiding pressor resuscitation and using sedatives, and performing therapies for reducing TBI.

[0080] That is, the present invention enables a subject with TBI to be quickly classified as mTBI or m-sTBI, so that the subject can receive the most appropriate treatment.

[0081] According to a further aspect of the present invention, there is provided a detection system for diagnosing and / or monitoring TBI, comprising a sensor element including a substrate functionalized with a probe specific for a target miRNA. The detection system can further comprise a detection device capable of detecting the binding of the target miRNA to the probe.

[0082] According to yet a further aspect of the present invention, there is provided a sensor element for use in a detection system for diagnosing and / or monitoring TBI, comprising a substrate functionalized with a probe specific for a target miRNA.

[0083] The sensor element can further include a sample addition zone for receiving a sample (e.g., a body fluid sample) thereon.

[0084] The probe is capable of selectively binding to the miRNA of interest. The substrate can be functionalized with a plurality of probes. The probes can all be the same or can provide two or more different probes. For example, in some embodiments, the substrate can be functionalized with a first probe specific for a first miRNA and a second probe specific for a second miRNA. The first and second probes can be grouped together, for example, on different portions of the sensor element.

[0085] In a further aspect of the present invention, there is provided a composition for use in a method of diagnosing and / or monitoring traumatic brain injury (TBI) in a subject, comprising a probe specific for a target miRNA. The composition can comprise any one of the listed miRNAs or a plurality of any of the listed miRNAs (e.g., two, three, four, or more of the listed miRNAs).

[0086] In some embodiments, the target miRNA is selected from the group consisting of miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-335, miR-126 * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 * , miR-671-3p, hsa-let-7c-5p, hsa-let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p, miR-221-3p, miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p, miR-30a-5p; miR-107; miR-135b-5p; miR-199b-5p; miR-324-5p; miR-652-3p, miR-424-5p, miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p and miR-629

[0087] In some embodiments, the target miRNA is miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-335, hsa-miR-126 * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 *Selected from the group consisting of, and miR-671-3p. These microRNAs have been found to be biomarkers expressed in all TBI patients (mild or severe).

[0088] In some embodiments, the target miRNA is selected from the group consisting of the following miRNAs: miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g and miR-335.

[0089] In some embodiments, the target miRNA is selected from the group consisting of let-7c-5p, let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p, miR-221-3p, miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p, and miR-424-5p; miR-30a-5p; miR-107; miR-135b-5p; miR-199b-5p; miR-324-5p; miR-652-3p.

[0090] In some embodiments, the target miRNA is selected from the group consisting of miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-194, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p and miR-629.

[0091] In some embodiments, the target miRNA is selected from the group consisting of the following miRNAs: miR-425-5p, miR-502, miR-21 and miR-335.

[0092] The probe can contain biological molecules such as proteins (e.g., antibodies) or nucleic acids. In some embodiments, the probe contains a nucleic acid. The nucleic acid can contain a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to a sequence that is complementary to the full-length sequence of the target miRNA. In some embodiments, the nucleic acid contains a sequence that is 100% identical to a sequence that is complementary to the sequence of the target miRNA (i.e., the receptor contains a nucleic acid sequence that is the exact complement of the target miRNA sequence).

[0093] The probe can be linked to the surface of the substrate by any suitable means, such as by coupling chemistry known to those skilled in the art. In some embodiments, each probe is linked to the surface of the substrate via a linker. In some embodiments, the probe contains a moiety for immobilizing the probe on the substrate or for linking the probe to a linker immobilized on the substrate.

[0094] Alternatively, or in addition thereto, the probe can contain a detectable label. The detectable label can be, for example, radioactive, fluorescent, luminescent, or antibody-based (e.g., can constitute a conventional tetrameric antibody or a detectable fragment thereof).

[0095] The substrate of the sensor element can be formed from any suitable material. In some embodiments, the substrate comprises or is formed from metal, plastic, glass, silica, silicon, graphite, graphene, or any combination thereof. In some embodiments, the substrate contains multiple layers. For example, the substrate can be fabricated by forming a graphene surface or layer on a layer of silicon carbide or silica. The graphene surface can be chemically modified, for example, to graphene oxide (GO) or graphene-amine (GA). Methods for forming graphene layers, such as epitaxial growth and sublimation growth, will be known to those skilled in the art.

[0096] Conventionally, a probe containing or composed of nucleic acid can be linked to the GO surface via a linker using an amino coupling reagent (e.g., O-(7-azabenzotriazol-1-yl)-N,N,N,N'-tetramethyluronium hexafluorophosphate (HATU)). A sensor element comprising a surface functionalized with a nucleic acid probe can subsequently be used to selectively detect the complementary miRNA thereto.

[0097] Suitable linkers can include an aniline moiety (or a derivative thereof), a benzoic acid moiety (or a derivative thereof), or an ethylenediamine moiety (or a derivative thereof). An aniline linker can be formed by linking a nitrobenzene molecule (or derivative) to the graphene surface (e.g., using a diazonium salt), and reducing the nitrobenzene to aniline. Subsequently, the amine group of the aniline can be used to link to the probe. Similarly, a diazonium salt (e.g., 4-benzoic acid diazonium tetrafluoroborate) can be used to link benzoic acid or a benzoic acid derivative to the graphene surface. The ethylenediamine moiety can be linked to carboxylated graphene or oxidized graphene.

[0098] The sensor element can be included within a test strip. The test strip can be disposable.

[0099] The detection device can be configured to detect the binding of the target miRNA to the receptor by any suitable means known to those skilled in the art, e.g., by detecting a change in electrical impedance, hydrogen ion concentration, or conformational change caused by hybridization.

[0100] The detection device can further comprise a user interface for outputting data to the user.

[0101] In some embodiments, the detection device comprises a database of treatment information. The device may be able to identify suitable treatment options from the database according to the miRNA of interest or the level of each miRNA. The treatment information can be provided to the user via a user interface.

[0102] Conveniently, the detection device can be portable, for example, handheld. The detection device can comprise a data storage unit for storing miRNA levels and other information regarding the subject. In some embodiments, the device includes data communication means for communicating data to other devices. For example, the device can communicate data wirelessly via WiFi, 3G, 4G, Bluetooth®, or other mobile applications. This can conveniently enable easy access to the data by medical professionals if necessary.

[0103] That is, the detection device of the present invention is expected to be affordable, portable, and provide a point of care (POC) means for non-invasively diagnosing and monitoring TBI. The device can be used by emergency responders, the military, schools, sports clubs, and medical professionals, thereby enabling accurate assessment and triage of patients suspected of having TBI.

[0104] In a further aspect, a kit for use in the present method is provided. The kit can comprise at least a probe (e.g., a protein such as an antibody, or a nucleic acid) capable of selectively binding to a target miRNA. In some embodiments, the kit comprises an array containing a plurality of probes. In some embodiments, at least one probe is a primer for performing PCR. The kit can further comprise instructions for use, such as a handling instruction for use in the diagnosis and / or monitoring of TBI. The kit can further comprise suitable buffers and reagents, such as amplification primers and enzymes (e.g., DNA polymerase, reverse transcriptase for the conversion of miRNA to cDNA).

[0105] It will be understood that any description made herein in connection with any aspect of the present invention can be equally applicable to any other aspect of the present invention, where appropriate. The present invention includes, for example, the following embodiments: [Embodiment 1] A method for diagnosing and / or monitoring traumatic brain injury (TBI) in a subject, comprising the step of determining the level of at least one miRNA in a body fluid sample derived from the subject. [Embodiment 2] The at least one miRNA is the following miRNAs: miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-335, miR-126 * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 *, miR-671-3p, hsa-let-7c-5p, hsa-let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p, miR-221-3p, miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p, miR-424-5p, miR-30a-5p; miR-107; miR-135b-5p; miR-199b-5p; miR-324-5p; miR-652-3p; miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p and miR-629, or any combination thereof The method according to embodiment 1, selected from the group consisting of [Embodiment 3] The at least one miRNA is the following miRNAs: miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-335, hsa-miR-126 * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 * , and miR-671-3p, or any combination thereof, the method according to embodiment 1 or 2 [Embodiment 4] The method according to any one of Embodiments 1 to 3, wherein the at least one miRNA is selected from the group consisting of the following miRNAs: miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, and miR-335, or any combination thereof. [Embodiment 5] The method according to Embodiment 1 or 2, wherein the at least one miRNA is selected from the group consisting of the following miRNAs: let-7c-5p, let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p, miR-221-3p, miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p, and miR-424-5p; miR-30a-5p; miR-107; miR-135b-5p; miR-199b-5p; miR-324-5p; miR-652-3p, or any combination thereof. [Embodiment 6] The method according to Embodiment 1 or 2, wherein the at least one miRNA is selected from the group consisting of the following miRNAs: miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-194, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p, and miR-629, or any combination thereof. [Embodiment 7] The method according to any one of Embodiments 1 to 4, wherein the at least one miRNA is selected from the group consisting of the following miRNAs: miR-425-5p; miR-502; miR-21; and miR-335, or any combination thereof. [Embodiment 8] The method according to embodiment 7, wherein the TBI is mild TBI (mTBI), and the miRNA is selected from the group consisting of the following miRNAs: miR-425-5p and miR-502. [Embodiment 9] The method according to embodiment 8, wherein when the level of miR-425-5p and / or miR-502 is determined to be less than a predetermined threshold or is decreased as compared with a reference, the subject is diagnosed as having mTBI. [Embodiment 10] The method according to embodiment 7, wherein the TBI is severe TBI (sTBI), and the miRNA is selected from the group consisting of the following miRNAs: miR-21 and miR-335. [Embodiment 11] The method according to embodiment 10, wherein when the levels of miR-21 and miR-335 are determined to exceed a predetermined threshold or are increased as compared with a reference, the subject is diagnosed as having sTBI. [Embodiment 12] The following miRNAs: (i) a first miRNA selected from miR-425-5p and miR-502; and (ii) a second miRNA selected from miR-21 and miR-335 The method according to embodiment 7, comprising the step of determining the levels of. [Embodiment 13] When the level of miR-425-5p or miR-502 is determined to be less than a predetermined threshold or is decreased as compared with a reference, and the level of miR-21 or miR-335 is determined to exceed a predetermined threshold or is increased as compared with a reference, the subject is diagnosed as having TBI of any severity. The method according to embodiment 12. [Embodiment 14] The method according to any one of embodiments 1 to 13, wherein the method is for diagnosing TBI, and the level of the at least one miRNA is determined in a body fluid sample obtained from the subject within 48 hours after the injury. [Embodiment 15] The method according to any one of embodiments 1 to 14, wherein the body fluid sample includes saliva, blood, plasma or serum. [Embodiment 16] The method according to Embodiment 15, wherein the body fluid sample is saliva. [Embodiment 17] A sensor element for a detection system for diagnosing and / or monitoring TBI, comprising a substrate functionalized with a probe specific for a target miRNA. [Embodiment 18] The sensor element according to Embodiment 17, wherein the probe contains nucleic acid. [Embodiment 19] The sensor element according to Embodiment 18, wherein the nucleic acid contains a sequence that is at least 80% identical to a sequence that is complementary to the sequence of the target miRNA. [Embodiment 20] The sensor element according to any one of Embodiments 17 to 19, wherein the substrate is formed from metal, plastic, glass, silica, silicon, graphite or graphene, or any combination thereof. [Embodiment 21] The following components: · A sensor element according to any one of Embodiments 17 to 20; and · A detection system for diagnosing and / or monitoring TBI, comprising a detection device capable of detecting the binding of a target miRNA to the probe. [Embodiment 22] A kit for use in a method of diagnosing and / or monitoring traumatic brain injury (TBI) in a body fluid sample derived from a subject, comprising at least one probe specific for a target miRNA. [Embodiment 23] The following steps: Determining the level of at least one miRNA in a body fluid sample derived from a subject; and Determining whether it is appropriate to administer a therapy for reducing TBI based on the level of the at least one miRNA A method for determining whether it is appropriate to administer a therapy for reducing TBI to a subject, comprising the above steps. [Embodiment 24] A method for determining appropriate treatment for a subject suspected of suffering from TBI, comprising identifying whether the subject has TBI by determining the level of at least one miRNA in a body fluid sample derived from the subject. [Embodiment 25] When the subject is identified as having TBI, an appropriate treatment is determined to be at least one of the following treatments: further evaluating the subject; excluding the subject from activities; admitting the subject to a hospital or outpatient clinic; and administering to the subject a therapy for reducing TBI, according to the method of Embodiment 24. [Embodiment 26] A therapy for reducing TBI for use in a method of treating a subject in need thereof, the therapy comprising determining the level of at least one miRNA in a body fluid sample from the subject, whereby the subject is identified as having TBI. [Embodiment 27] The therapy for reducing TBI is selected from at least one of the following therapies: neuroprotective agents, sedatives; and avoidance of the use of hypertensive resuscitation, according to any of Embodiments 24 to 26. [Embodiment 28] A composition for use in a method of diagnosing and / or monitoring traumatic brain injury (TBI) in a subject, the composition comprising a probe specific for a target miRNA. [Embodiment 29] The composition according to Embodiment 28, comprising a plurality of probes. [Embodiment 30] The miRNA is one of the following miRNAs: miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-335, miR-126 * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 *, miR-671-3p, hsa-let-7c-5p, hsa-let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p, miR-221-3p, miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p, miR-30a-5p; miR-107; miR-135b-5p; miR-199b-5p; miR-324-5p; miR-652-3p, miR-424-5p, miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p, and miR-629, or any combination thereof, a sensor element, detection system, kit, method, or composition according to any of embodiments 17 to 29. [Embodiment 31] The miRNA is selected from the group consisting of the following miRNAs: miR-425-5p; miR-502; miR-21; and miR-335, or any combination thereof, a sensor element, detection system, kit, method, or composition according to embodiment 30.

[0106] Detailed Description of the Invention Embodiments of the present invention will now be described by way of example and with reference to the accompanying drawings.

[0107] Figure 1: Diagram showing miR-425-5p and miR-502 expression in three different classifications of trauma and healthy volunteers (HV). miR-425-5p and miR-502 expression in 10 HV, 10 mild traumatic brain injury + early concussion (mTBI+EC, 1 day), 10 mTBI+EC (15 days), 10 early concussion (EC, 1 day), 10 EC (15 days), 10 severe traumatic brain injury + early concussion (sTBI+EC, 1 day), and 10 sTBI+EC (15 days) patients detected by qRT-PCR analysis. miR-425-5p expression was found to be significantly decreased in mTBI+EC (1 day) compared to HV (p<0.01), mTBI+EC (15 days) (p<0.001), and sTBI+EC (1 day) (p<0.01) (Figure 1A). miR-502 expression was found to be significantly decreased in mTBI+EC (1 day) compared to HV (p<0.05), mTBI+EC (15 days) (p<0.01), and sTBI+EC (1 day) (p<0.05) (Figure 1B).

[0108] Figure 2: Diagram showing miR-21 and miR-335 expression in three different classifications of trauma and healthy volunteers (HV). miR-21 and miR-335 expression in 10 HV, 10 mild traumatic brain injury + early concussion (mTBI+EC, 1 day), 10 mTBI+EC (15 days), 10 early concussion (EC, 1 day), 10 EC (15 days), 10 severe traumatic brain injury + early concussion (sTBI+EC, 1 day), and 10 sTBI+EC (15 days) patients detected by qRT-PCR analysis. miR-21 expression was found to be significantly upregulated in sTBI+EC (1 day and 15 days) compared to HV (p<0.01) (Figure 2A). miR-335 expression was found to be significantly upregulated in sTBI+EC (1 day) compared to HV (p<0.001), EC (15 days) (p<0.001), and mTBI+EC (1 day) (p<0.05) (Figure 2B).

[0109] Figure 3A: This is a diagram showing the time course of miR-425-5p expression in three different classifications of trauma and healthy volunteers (HV). miR-425-5p expression at various time points (T0, T4 - 12 h, T48 - 72 h, 15 days) after injury in 30 HV, 30 mild traumatic brain injury with extracranial injury (mTBI+EC), 30 extracranial injury (EC), and 30 severe traumatic brain injury with extracranial injury (sTBI+EC) patients detected by qRT-PCR analysis. miR-425-5p expression was found to be significantly decreased in mTBI+EC (T0 and T4 - 12 h) compared to HV, sTBI+EC, and EC (p < 0.05). The p-value was determined by Tukey's post hoc test. *: Significantly different from HV.

[0110] Figure 3B: This is a diagram showing the time course of miR-502 expression in three different classifications of trauma and healthy volunteers (HV). miR-502 expression at various time points (T0, T4 - 12 h, T48 - 72 h, 15 days) after injury in 30 HV, 30 mild traumatic brain injury with extracranial injury (mTBI+EC), 30 extracranial injury (EC), and 30 severe traumatic brain injury with extracranial injury (sTBI+EC) patients detected by qRT-PCR analysis. miR-502 expression was found to be significantly decreased in mTBI+EC (T0 and T4 - 12 h) compared to HV, sTBI+EC, and EC (p < 0.05). The p-value was determined by Tukey's post hoc test. *: Significantly different from HV.

[0111] Figure 4A: This is a diagram showing the time course of miR-21 expression in three different classifications of trauma and healthy volunteers (HV). miR-21 expression at various time points (T0, T4 - 12 h, T48 - 72 h, 15 days) after injury in 30 HV, 30 mild traumatic brain injury with extracranial injury (mTBI+EC), 30 extracranial injury (EC), and 30 severe traumatic brain injury with extracranial injury (sTBI+EC) patients detected by qRT-PCR analysis. miR-21 expression was found to be significantly upregulated in sTBI+EC (T4 - 12 h, T48 - 72 h, and 15 days) compared to HV (p < 0.01). The p-value was determined by Tukey's post hoc test. *: Significantly different from HV.

[0112] Figure 4B: A diagram showing the time course of miR-335 expression in three different categories of trauma and HV. miR-335 expression at various time points (T0, T4-12h, T48-72h, 15 days) from injury in 30 HV, 30 mTBI+EC, 30 EC, and 30 sTBI+EC patients detected by qRT-PCR analysis. miR-335 expression was found to be significantly upregulated in sTBI+EC (T0, T4-12h, T48-72h and 15 days) compared to HV and mTBI+EC (p<0.001), but not significantly upregulated compared to EC only. p-values were determined by Tukey's post hoc test. *: Significantly different from HV.

Example

[0113] Since miRNAs have been found to be promising biomarkers in a variety of pathological conditions within a certain range, the present inventors have attempted to explore their roles in TBI.

[0114] Example 1 Materials and Methods Patients and Sample Collection Participants in the study were recruited from the Surgical Reconstruction and Microbiology Research Centre (SRMRC) at Queen Elizabeth Hospital, Birmingham (UK) as part of a study on post-traumatic brain biomarkers (the Golden Hour study) (Ethics Ref. 13 / WA / 0399).

[0115] First, the inventors selected 5 mTBI patients with extra-cranial injury (EC), 5 sTBI+EC injury patients, and healthy volunteers (HV) to identify specific candidate biomarkers that can distinguish mild TBI from severe TBI and predict the recovery of mTBI after 15 days. Screening of 754 miRNAs was performed at 1 day and 15 days after injury. Based on this information (Table 2), it was then possible to confirm the results of a study in an expanded cohort of 40 patients divided into the following 4 different classifications: HV (n = 10), EC (n = 10), mTBI+EC (n = 10), sTBI+EC (n = 10). Healthy volunteers consented to participate in the RECOS study. EC injury patients had fractures confirmed by radiography, no head injury, no history of infectious disease, neurological disorder or psychiatric disorder, and no alcohol or drug dependence. Mild TBI with EC included patients with non-penetrating head injury and a Glasgow Coma Scale (GCS) score >13. Severe TBI with EC included patients with a GCS score of 8 or less. All patients were matched for gender and age to the HVs.

[0116] Sample Processing Peripheral blood samples were obtained from each patient at 1 day and 15 days after injury. Blood samples were processed for serum separation within 2 hours after blood collection. Whole blood was allowed to stand at room temperature for 30 minutes and then centrifuged at 3000 rpm for 10 minutes at 4°C. Serum was aliquoted and stored at -80°C until analysis.

[0117] RNA Isolation, Reverse Transcription, and miRNA Profiling by TaqMan Low Density Array (TLDA) Initial screening (discovery set) was performed on 5 mTBI+EC patients and 5 sTBI+EC patients, and compared with HV at two different time points (1 day and 15 days after injury). Using the sera of these patients, transcriptome profiles of 754 miRNAs were created. Serum samples were centrifuged at 2000 rpm for 10 minutes to pellet, and circulating cells or residues were removed. miRNAs were extracted from 400 μL of serum samples by using the Qiagen miRNeasy mini kit (Qiagen GmbH, Hilden, Germany) according to Qiagen's supplementary protocol for the purification of small RNAs from serum and plasma, and finally eluted in a 30 μL volume of RNase-free water. The concentration and purity of the obtained RNA were determined using an ND-1000 UV-Vis spectrophotometer (NanoDrop). 20 ng of serum RNA was retrotranscribed and pre-amplified according to the manufacturer's instructions. The pre-amplified products were loaded onto a TLDA (TaqMan Human MicroRNA array v3.0 A and B (Applied Biosystems LifeTechnologies TM ))). PCR on the TLDA was performed using a 7900HT Fast RealTime PCR system (Applied Biosystem, LifeTechnologies TM ).

[0118] Data Analysis To obtain accurate miRNA profiles, the inventors used a global median normalization method. Similar to microarray analysis, the Ct values from each sample were normalized to the median Ct of the array. Furthermore, by calculating the Pearson correction between the Ct median and mean of each array and the Ct of each miRNA, the inventors identified two miRNAs, miR-331 and miR-223, that showed expression profiles approximated to the median and mean of the TLDA *were identified. These miRNAs were also confirmed to be among the most stable in the TLDA by applying two different methods: DataAssist v.3 software (Applied Biosystem Life Technologies TM ). Thus, miR-331 and miR-223 * were used as reference genes for validation by single TaqMan assay. The fold change in expression was calculated by the 2-ΔΔCT method. Differentially expressed miRNAs (DE miRNAs) were identified by Significance of Microarrays Analysis (SAM) using the Multi Experiment Viewer v4.8.1, applying a two-class unpaired test for the correspondence between ΔCt and using a p-value based on 100 permutations; Complementary engine: K-nearest neighbor (10 neighbors); False discovery rate <0.15 was used as a correction for multiple comparisons. The inventors found that only DE miRNAs were highly reliable, consistent with using all endogenous controls.

[0119] Single TaqMan Assay For the purpose of differentiating mild TBI from severe TBI and monitoring the recovery of mild TBI, 10 differentially expressed miRNAs were selected from the array as potential candidate biomarkers. Using these candidates, data were validated in an expanded cohort of 30 patients (validation set) and 10 controls (HV) divided into three different classifications (mTBI+EC, sTBI+EC, and EC only) at two different time points (1 day and 15 days after injury) by a single TaqMan assay (Applied Biosystems, Life Technologies TM ). As described above, samples were extracted and reverse transcribed, and RT-qPCR analysis was performed using a Bio-Rad iQ5 Real-time PCR Detection system (Bio-Rad, CA, USA). The fold change in expression was calculated by the 2-ΔΔCT method.

[0120] Statistical Analysis The data were checked for normality and transformed for parametric testing. Comparisons between groups at each time point and within groups over time were performed on the transformed data by one-way analysis of variance and Tukey's post hoc test. The sensitivity and specificity of each biomarker for the diagnosis of either mTBI or sTBI, expressed as the area under the curve (AUC), were calculated using receiver operating characteristic analysis. All analyses were performed using SPSS v.20 (IBM). Differences were considered statistically significant if the p-value was less than 0.05.

[0121] Results Expression Profile by TaqMan Low Density Array (TLDA) From the 754 screenable miRNAs of TLDA, the inventors identified 10 circulating miRNAs (day 1) and 13 (day 15) as differentially expressed in mTBI+EC, and 19 (day 1) and 22 (day 15) as differentially expressed in sTBI+EC (Table 2). From this list, hsa-miR-126 * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 * and miR-671-3p were excluded from further analysis as they were expressed in the majority of patients and thus not suitable candidate biomarkers for mild or severe trauma only. However, the above microRNAs can be identified in TBI of any severity and are therefore useful TBI biomarkers. On the other hand, miR-184, miR-301b, miR-502 and miR-505 were specifically and differentially expressed in mTBI+EC (day 1) and were selected as initial candidate biomarkers for mTBI. In addition, miR-203, miR-425-5p, miR-654-3p and miR-655, which were differentially expressed 15 days after mTBI+EC, were selected as candidate biomarkers for tracking the recovery of mTBI.

[0122] Finally, miR-21 and miR-335, two miRNAs that are constantly expressed at both time points in sTBI+EC, were selected for further study.

[0123]

Table 2

[0124] Single TaqMan Assay for Candidate Biomarkers of mTBI To verify these findings, the inventors then examined the expression of the selected miRNAs in three separate independent groups (10 mTBI+EC, 10 sTBI+EC, 10 EC) at two selected time points (1 day and 15 days after injury) using a single TaqMan assay. The results were compared to 10 HV. The fold change was calculated by the 2-ΔΔCT method using miR-331 and miR-223 * as reference genes.

[0125] Among the candidate biomarkers for mTBI (miR-184, miR-301b, miR-502, miR-505, miR-203, miR-425-5p, miR-654-3p, and miR-655) at both time points, two showed interesting results and were significantly and differentially expressed in three different classifications compared to HV. In particular, miR-425-5p and miR-502 showed similar trends (Figure 1). Both were significantly downregulated in mTBI+EC at 1 day post-injury (mean 0.387±0.201 and 0.314±0.146) compared to HV (p<0.001), EC (p<0.001), and sTBI+EC (p<0.001), respectively. At 15 days post-mild injury, miR425-5p and miR-502 returned to normal levels (0.886±0.310 and 1.157±0.258). The expression of miR-425-5p and miR-502 was also found to be similar to HV in EC samples at 1 and 15 days post-injury, suggesting that these two biomarkers are differentially expressed only in patients with brain injury. Furthermore, none of them showed a significant difference in sTBI+EC compared to HV at both time points. Therefore, miR-425-5p and miR-502 could be considered the most promising candidate biomarkers for the early diagnosis and monitoring of mTBI at 15 days post-injury. The AUC for these biomarkers is shown in Table 3.

[0126] Single TaqMan Assay for Candidate Biomarkers of sTBI Since both of them were found to be upregulated at both time points of sTBI+EC in the initial screening, miR-21 and miR-335 were analyzed as potential biomarkers for sTBI. These were also shown to be strong candidates in the second patient dataset (Figure 2). miR-21 was significantly upregulated (7.106±4.192 and 4.012±1.577) at both time points in sTBI with EC, against HV (p, 0.001), EC (p<0.001) and mTBI (p<0.001). No significant difference was found in the residual classification compared with HV. miR-335 showed upregulation at both time points in sTBI+EC (16.824±14.195 and 12.324±8.931, respectively). On day 1, these groups were significantly different from the control (p=0.001) and mTBI+EC (p=0.031), but not significantly different from EC. Interestingly, significant upregulation in EC patients was seen on day 1 (7.951±4.870), but not 15 days after injury (1.260±0.531). For this reason, on day 15, miR335 was significantly higher in the sTBI+EC group against HV (p=0.002), EC (p=0.007) and mTBI+EC (p=0.001). miR-335 showed no significant difference at both time points in mTBI+EC compared with HV. The AUCs for these biomarkers are also shown in Table 3.

[0127]

Table 3

[0128] Discussion In this study, we investigated whether changes at the miRNA level could be applied to the diagnosis of TBI and the assessment of its severity. Four miRNAs were identified as differentially expressed in TBI: miR-425-5p, miR 502, miR-21 and miR-335.

[0129] Compared with HV, miR-425-5p showed significant results on the first day in mTBI+EC, and similar results were obtained in all other classifications. Due to its downregulation within the first 24 hours after mild injury and its return to normal levels 15 days later, miR-425-5p is a suitable candidate biomarker for mild trauma.

[0130] MiR-502 was also found to be differentially expressed in mTBI+EC. The trend of this miRNA was very similar to that of miR-425-5p. Since this miRNA shows specificity for brain injury patients and returns to normal values 15 days after mild injury, it could also be used to track recovery.

[0131] After sTBI, two miRNAs (miR-21, miR-335) were noted to be expressed at both 1 day and 15 days, that is, they were selected as potential biomarkers for sTBI. miR-21 and miR-335 were significantly upregulated at both time points when compared to the control in sTBI+EC. Thus, the overexpression confirmed the array results and indicated the potential of these molecules as biomarkers for sTBI.

[0132] The selected panel of miRNAs has the ability to accurately diagnose TBI and enable the stratification of patients according to its severity, thus enabling the provision of the most appropriate treatment.

[0133] Example 2 Patients and Sample Collection Participants in the study were recruited from the Surgical Reconstruction and Microbiology Research Centre (SRMRC), Queen Elizabeth Hospital, Birmingham (UK) as part of the SIR (Steroids and Immunity from injury through to Rehabilitation) study (Ethics Ref. 11 / SW / 0177), the ReCoS (REpetitive COncussion in Sport) study (Ethics Ref. 11-0429AP28) and the Golden Hour study (Ethics Ref. 13 / WA / 0399). Written informed consent was obtained from participants or a legally valid proxy (family member or expert not directly involved in the study) prior to incorporation into the study.

[0134] The second sample dataset used serum samples from a total of 120 individuals divided into four different classifications: HV (n = 30), EC (n = 30), mTBI+EC (n = 30), sTBI+EC (n = 30). For each patient, blood was taken at different time points (T0 - 1h, T4 - 12h, T48 - 72h, 15 days). Healthy volunteers consented to participate in the ReCoS study. EC injury patients had orthopaedic fractures confirmed by radiography, no head injury, no history of infection, neurological or psychiatric disorder, and no alcohol or drug dependence. Mild TBI with EC included patients with non-penetrating head injury and a Glasgow Coma Scale (GCS) score ≥13. Severe TBI with EC included patients with GCS ≤8.

[0135] Sample Processing Blood samples were processed for serum separation within 2 hours of collection. Whole blood was left to stand at room temperature for approximately 30 minutes, followed by centrifugation at 3000 rpm for 10 minutes at 4°C. Serum was aliquoted and stored at -80°C until analysis.

[0136] RNA isolation, data analysis, assays, and statistical analysis were performed as described in Example 1.

[0137] Results Single TaqMan Assay for Candidate Biomarkers of mTBI To validate the findings in Example 1, the expression of selected miRNAs in three separate independent groups (30 mTBI + EC, 30 sTBI + EC, 30 EC) was measured at various time points (T0, T4 - 12 h, T48 - 72 h, and 15 days post-injury) using a single TaqMan assay. Results were compared to 10 HV. Fold changes were calculated by the 2-ΔΔCT method using miR-331 and miR-223 * as references.

[0138] Among the candidate biomarkers for mTBI (miR-184, miR-502, miR-505, miR-301b, miR-203, miR-425-5p, miR-654-3p, and miR-655) at both time points, only two showed interesting results and were significantly and differentially expressed in three different classifications compared to HV. Specifically, miR-425-5p and miR-502 showed similar trends (Figure 3). Both were downregulated in mTBI+EC, and compared to HV or EC and sTBI+EC (p<0.05), miR-425-5p was downregulated at T0~1h (p=0.01845) and T4-12h (p=0.01962), respectively, and miR-502 was downregulated at T0~1h and T4~12h compared to HV (p=0.02538 and p=0.03718, respectively), or compared to EC and sTBI+EC (p<0.01). Forty-eight hours after mild injury, miR425-5p and miR-502 returned to normal levels. The expression of miR-425-5p and miR-502 was also found at levels equivalent to HV in the EC group, suggesting that these two biomarkers are downregulated only in patients with brain injury. Furthermore, none of these showed significant downregulation in sTBI+EC compared to HV at all time points. Therefore, miR-425-5p and miR-502 could be considered the most promising candidate biomarkers for the early diagnosis and monitoring of mTBI. The AUC for these biomarkers at the most important time points is shown in Table 4.

[0139] Single TaqMan Assay for Candidate Biomarkers of sTBI Both miR-21 and miR-335 were analyzed as potential biomarkers for sTBI since both were found to be upregulated at both time points of sTBI+EC in the initial screening. These were also shown to be strong candidates in the second patient dataset (Figure 4). miR-21 was significantly upregulated in sTBI with EC at all time points 4 hours after injury compared to HV, EC, and mTBI+EC (p = 0.00306 (T4~12h), p = 0.00844 (T48~72h), and p = 0.00077 (15 days)). No significant difference was found in the remaining classifications compared to HV. miR-335 showed upregulation at all time points in sTBI+EC compared to HV (p = 0.00109 (T0~1h), p = 0.00284 (T4~12h), p = 0.00012 (T48~72h), and p = 0.01284 (15 days)) and showed upregulation in mTBI+EC, but no significant upregulation was found compared to EC. The AUCs for these biomarkers are also shown in Table 4.

[0140]

Table 4

[0141] Discussion In this study, we verified the previous finding that changes in miRNA levels can be applied to the diagnosis of TBI and the assessment of its severity. This study confirmed that the following four miRNAs are differentially expressed in TBI: miR-425-5p, miR 502, miR-21, and miR-335.

[0142] miR-425-5p showed significant results at T0 and T4~12h in mTBI+EC compared to HV, and similar results were obtained in all other classifications. Its downregulation returned to normal levels after T48~72h, confirming that miR-425-5p is a suitable candidate biomarker for mild trauma.

[0143] MiR-502 was also confirmed to be differentially expressed in mTBI+EC. The trend of this miRNA was very similar to that of miR-425-5p. This miRNA shows specificity for brain injury patients and returns to normal values 48 - 72 hours after mild injury, so it could also be used to track recovery.

[0144] After sTBI, two miRNAs (miR-21, miR-335) were noted to be expressed at all time points analyzed, that is, they were identified as potential biomarkers for sTBI. miR-21 and miR-335 were significantly upregulated in sTBI+EC when compared to the control. Thus, overexpression confirmed the potential of these molecules as biomarkers for sTBI.

[0145] Example 3 Saliva samples were collected from professional sports players who had suffered concussion 2 - 3 days after injury, and the microRNAs present in the saliva were analyzed. The sports players were clinically diagnosed with mTBI.

[0146] Materials and Methods MicroRNAs were analyzed using the nCounter technology (nanoString Technologies®), which uses molecular "barcodes" and microscopy imaging to detect and count up to hundreds of unique transcripts in a single hybridization reaction. Each color-coded barcode is linked to a single target-specific probe corresponding to the microRNA of interest.

[0147] The analysis was performed according to the manufacturer's protocol, which included the following steps:

[0148] Hybridization: This technique utilizes two approximately 20-base probes per microRNA that hybridize in solution. The reporter probe retains the signal and the capture probe enables the complex to be immobilized for data collection. Purification and Immobilization : After hybridization, excess probes are removed and the probe / target complex is aligned and immobilized in the nCounter cartridge. Data Collection : The sample cartridge is placed in a digital analyzer device for data collection. The color code on the surface of the cartridge is counted and tabulated for each target molecule.

[0149] Results The following Table 5 is a list of microRNAs that were found to be significantly and differentially expressed in sports players with concussion compared to healthy volunteers. This table shows the fold change in microRNA expression in patients compared to the control group. The fold change was calculated using miR-23a-3p and miR-148b-3p as reference genes.

[0150]

Table 5

[0151] Discussion This study shows that microRNAs present in saliva are indicators of concussion / mTBI. This is important because saliva is more easily obtained than blood, meaning that detection of microRNAs in saliva provides a rapid and simple means of diagnosing TBI, particularly at the sidelines of the arena.

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Claims

1. A method of diagnosing and / or monitoring traumatic brain injury (TBI) in a subject comprising determining the level of at least one miRNA in a bodily fluid sample from the subject.

2. The at least one miRNA is selected from the group consisting of: miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-335, miR-126 * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 * , miR-671-3p, hsa-let-7c-5p, hsa-let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p, miR-221-3p, miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p, miR-424-5p, miR-30a-5p; miR-107; miR-135b-5p; miR-199b-5p; miR-324-5p; miR-652-3p; miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p and miR-629, or any combination thereof 2. The method of claim 1, selected from the group consisting of:

3. The at least one miRNA is selected from the group consisting of miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-335, and hsa-miR-126. * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 * 3. The method of claim 1 or 2, wherein the IL-11A1-binding domain is selected from the group consisting of miR-671-3p, and miR-671-3p, or any combination thereof.

4. 4. The method of any one of claims 1 to 3, wherein the at least one miRNA is selected from the group consisting of the following miRNAs: miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, and miR-335, or any combination thereof.

5. 3. The method of claim 1 or 2, wherein the at least one miRNA is selected from the group consisting of the following miRNAs: let-7c-5p, let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p, miR-221-3p, miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p, and miR-424-5p; miR-30a-5p; miR-107; miR-135b-5p; miR-199b-5p; miR-324-5p; miR-652-3p, or any combination thereof.

6. 3. The method of claim 1 or 2, wherein the at least one miRNA is selected from the group consisting of the following miRNAs: miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-194, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p, and miR-629, or any combination thereof.

7. 5. The method of any one of claims 1 to 4, wherein the at least one miRNA is selected from the group consisting of the following miRNAs: miR-425-5p; miR-502; miR-21; and miR-335, or any combination thereof.

8. 8. The method of claim 7, wherein the TBI is mild TBI (mTBI) and the miRNA is selected from the group consisting of the following miRNAs: miR-425-5p and miR-502.

9. The method of claim 8, wherein the subject is diagnosed with mTBI when the levels of miR-425-5p and / or miR-502 are determined to be below a predetermined threshold or are decreased compared to a reference.

10. 8. The method of claim 7, wherein the TBI is severe TBI (sTBI) and the miRNA is selected from the group consisting of the following miRNAs: miR-21 and miR-335.

11. The method of claim 10, wherein the subject is diagnosed with sTBI when the levels of miR-21 and miR-335 are determined to exceed a predetermined threshold or are increased compared to a reference.

12. The following miRNAs: (i) a first miRNA selected from miR-425-5p and miR-502; and (ii) a second miRNA selected from miR-21 and miR-335; 8. The method of claim 7, further comprising determining a level of

13. The method of claim 12, wherein the subject is diagnosed as having any severity of TBI when the level of miR-425-5p or miR-502 is determined to be below a predetermined threshold or is decreased compared to the reference, and the level of miR-21 or miR-335 is determined to be above a predetermined threshold or is increased compared to the reference.

14. 14. The method of any one of claims 1 to 13, wherein the method is for diagnosing TBI and the level of the at least one miRNA is determined in a body fluid sample obtained from the subject no more than 48 hours after injury.

15. The method of any one of claims 1 to 14, wherein the body fluid sample comprises saliva, blood, plasma or serum.

16. The method of claim 15, wherein the bodily fluid sample is saliva.

17. A sensor element for a detection system for diagnosing and / or monitoring TBI, comprising a substrate functionalized with a probe specific for a target miRNA.

18. The sensor element of claim 17 , wherein the probe comprises a nucleic acid.

19. The sensor element of claim 18, wherein the nucleic acid comprises a sequence that is at least 80% identical to a sequence that is the complement of the sequence of the target miRNA.

20. The sensor element according to any one of claims 17 to 19, wherein the substrate is formed from metal, plastic, glass, silica, silicon, graphite or graphene, or any combination thereof.

21. Components: - a sensor element according to any one of claims 17 to 20; and A detection system for diagnosing and / or monitoring TBI, comprising a detection device capable of detecting binding of a target miRNA to said probe.

22. A kit for use in a method for diagnosing and / or monitoring traumatic brain injury (TBI) in a body fluid sample from a subject, comprising at least one probe specific for a target miRNA.

23. Steps below: Determining the level of at least one miRNA in a bodily fluid sample from the subject; and determining whether or not it is appropriate to administer a therapy to reduce TBI based on the level of the at least one miRNA; The present invention relates to a method for determining whether it is appropriate to administer to a subject a therapy to reduce TBI, comprising:

24. A method for determining an appropriate treatment for a subject suspected of suffering from TBI, comprising a step of identifying whether the subject has TBI by determining the level of at least one miRNA in a bodily fluid sample from the subject.

25. 25. The method of claim 24, wherein if the subject is identified as having a TBI, appropriate treatment is determined to be at least one of the following treatments: further evaluating the subject; removing the subject from activity; admitting the subject to a hospital or outpatient facility; and administering a therapy to the subject to reduce the TBI.

26. A therapy for reducing TBI for use in a method of treating a subject in need thereof, wherein the subject is identified as having TBI by determining the level of at least one miRNA in a bodily fluid sample from the subject.

27. 27. The method of any one of claims 24-26, wherein the therapy for reducing TBI is selected from at least one of the following therapies: neuroprotective drugs, sedatives; and avoidance of the use of hypertensive resuscitation.

28. A composition for use in a method of diagnosing and / or monitoring traumatic brain injury (TBI) in a subject, comprising a probe specific for a target miRNA.

29. 30. The composition of claim 28, comprising a plurality of probes.

30. The miRNA is the following miRNA: miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-335, miR-126 * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 * , miR-671-3p, hsa-let-7c-5p, hsa-let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p, miR-221-3p , miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p, miR-30a-5p; miR-107; m iR-135b-5p; miR-199b-5p; miR-324-5p; miR-652-3p, miR-424-5p, miR-10a, m 30. The sensor element, detection system, kit, method or composition of any one of claims 17 to 29, wherein the nucleic acid sequence is selected from the group consisting of iR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p and miR-629, or any combination thereof.

31. The sensor element, detection system, kit, method or composition of claim 30, wherein the miRNA is selected from the group consisting of the following miRNAs: miR-425-5p; miR-502; miR-21; and miR-335, or any combination thereof.

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