Mette Heiskanen

Objective: To test a hypothesis that acutely regulated plasma microRNAs (miRNAs) can serve as prognostic biomarkers for the development of post‐traumatic epilepsy (PTE). Methods: Adult male Sprague–Dawley rats ( n = 245) were randomized to lateral fluid‐percussion–induced traumatic brain injury (TBI) or sham operation at three study sites (Finland, Australia, United States). Video‐electroencephalography (vEEG) was performed on the seventh post‐injury month to detect spontaneous seizures. Tail vein plasma collected 48 h after TBI for miRNA analysis was available from 209 vEEG monitored animals (45 sham, 164 TBI [32 with epilepsy]). Based on small RNA sequencing and previous data, the seven most promising brain enriched miRNAs (miR‐183‐5p, miR‐323‐3p, miR‐434‐3p, miR‐9a‐3p, miR‐124‐3p, miR‐132‐3p, and miR‐212‐3p) were validated by droplet digital polymerase chain reaction (ddPCR). Results: All seven plasma miRNAs differentiated between TBI and sham‐operated rats. None of the seven miRNAs differentiated TBI rats that did and did not develop epilepsy ( p > .05), or rats with ≥3 vs .05). However, miR‐212‐3p differentiated rats that developed epilepsy with seizure clusters (i.e., ≥3 seizures within 24 h) from those without seizure clusters (.34 ± .14 vs .60 ± .34, adj. p < .05) with an area under the curve (AUC) of .81 (95% confidence interval [CI] .65–.97, p < .01, 64% sensitivity, 95% specificity). Lack of elevation in miR‐212‐3p also differentiated rats that developed epilepsy with seizure clusters from all other TBI rats ( n = 146, .34 ± .14 vs .55 ± .31, p < .01) with an AUC of .74 (95% CI .61–.87, p < .01, 82% sensitivity, 62% specificity). Glmnet analysis identified a combination of miR‐212‐3p and miR‐132‐3p as an optimal set to differentiate TBI rats with vs without seizure clusters (cross‐validated AUC .75, 95% CI .47–.92, p < .05). Significance: miR‐212‐3p alone or in combination with miR‐132‐3p shows promise as a translational prognostic biomarker for the development of severe PTE with seizure clusters.

Objective: This study was undertaken to test whether the postinjury plasma concentration of phosphorylated neurofilament heavy chain (pNF‐H), a marker of axonal injury, is a prognostic biomarker for the development of posttraumatic epilepsy.Methods: Tail vein plasma was sampled 48 h after traumatic brain injury (TBI) from 143 rats (10 naïve, 21 controls, 112 with lateral fluid percussion injury) to quantify pNF‐H by enzyme‐linked immunosorbent assay. During the 6th postinjury month, rats underwent 30 days of continuous video‐electroencephalographic monitoring to detect unprovoked seizures and evaluate epilepsy severity. Somatomotor (composite neuroscore) and spatial memory (Morris water maze) testing and quantitative T₂ magnetic resonance imaging were performed to assess comorbidities and lesion severity.Results: Of the 112 TBI rats, 25% (28/112) developed epilepsy (TBI+) and 75% (84/112) did not (TBI−). Plasma pNF‐H concentrations were higher in TBI+ rats than in TBI− rats (p < .05). Receiver operating characteristic curve analysis indicated that plasma pNF‐H concentration distinguished TBI+ rats from TBI− rats (area under the curve [AUC] = .647, p < .05). Differentiation was stronger when comparing TBI+ rats exhibiting severe epilepsy (≥3 seizures/month) with all other TBI rats (AUC = .732, p < .01). Plasma pNF‐H concentration on day 2 (D2) distinguished TBI+ rats with seizure clusters from other TBI rats (AUC = .732, p < .05). Higher plasma pNF‐H concentration on D2 after TBI correlated with lower neuroscores on D2 (p < .001), D6 (p < .001), and D14 (p < .01). Higher pNF‐H concentration on D2 correlated with greater T₂ signal abnormality volume on D2 (p < .001) and D7 (p < .01) and larger cortical lesion area on D182 (p < .01). Plasma pNF‐H concentration on D2 did not correlate with Morris water maze performance on D37–D39.Significance: Plasma pNF‐H is a promising clinically translatable prognostic biomarker for the development of posttraumatic epilepsy with frequent seizures or seizure clusters.

Traumatic brain injury (TBI) causes 10–20% of structural epilepsies and 5% of all epilepsies. The lack of prognostic biomarkers for post-traumatic epilepsy (PTE) is a major obstacle to the development of anti-epileptogenic treatments. Previous studies revealed TBI-induced alterations in blood microRNA (miRNA) levels, and patients with epilepsy exhibit dysregulation of blood miRNAs. We hypothesized that acutely altered plasma miRNAs could serve as prognostic biomarkers for brain damage severity and the development of PTE. To investigate this, epileptogenesis was induced in adult male Sprague Dawley rats by lateral fluid-percussion-induced TBI. Epilepsy was defined as the occurrence of at least one unprovoked seizure during continuous 1-month video-electroencephalography monitoring in the sixth post-TBI month. Cortical pathology was analyzed by magnetic resonance imaging on day 2 (D2), D7, and D21, and by histology 6 months post-TBI. Small RNA sequencing was performed from tail-vein plasma samples on D2 and D9 after TBI (n = 16, 7 with and 9 without epilepsy) or sham operation (n = 4). The most promising miRNA biomarker candidates were validated by droplet digital polymerase chain reaction in a validation cohort of 115 rats (8 naïve, 17 sham, and 90 TBI rats [21 with epilepsy]). These included 7 brain-enriched plasma miRNAs (miR-434-3p, miR-9a-3p, miR-136-3p, miR-323-3p, miR-124-3p, miR-212-3p, and miR-132-3p) that were upregulated on D2 post-TBI (p < 0.001 for all compared with naïve rats). The acute post-TBI plasma miRNA profile did not predict the subsequent development of PTE or PTE severity. Plasma miRNA levels, however, predicted the cortical pathology severity on D2 (Spearman ρ = 0.345–0.582, p < 0.001), D9 (ρ = 0.287–0.522, p < 0.001–0.01), D21 (ρ = 0.269–0.581, p < 0.001–0.05) and at 6 months post-TBI (ρ = 0.230–0.433, p < 0.001–0.05). We found that the levels of 6 of 7 miRNAs also reflected mild brain injury caused by the craniotomy during sham operation (ROC AUC 0.76–0.96, p < 0.001–0.05). In conclusion, our findings revealed that increased levels of neuronally enriched miRNAs in the blood circulation after TBI reflect the extent of cortical injury in the brain but do not predict PTE development.

Plasma neurofilament light chain (NF-L) levels were assessed as a diagnostic biomarker for traumatic brain injury (TBI) and as a prognostic biomarker for somatomotor recovery, cognitive decline, and epileptogenesis. Rats with severe TBI induced by lateral fluid-percussion injury (n = 26, 13 with and 13 without epilepsy) or sham-operation (n = 8) were studied. During a 6-month follow-up, rats underwent magnetic resonance imaging (MRI) (day (D) 2, D7, and D21), composite neuroscore (D2, D6, and D14), Morris-water maze (D35–D39), and a 1-month-long video-electroencephalogram to detect unprovoked seizures during the 6th month. Plasma NF-L levels were assessed using a single-molecule assay at baseline (i.e., naïve animals) and on D2, D9, and D178 after TBI or a sham operation. Plasma NF-L levels were 483-fold higher on D2 (5072.0 ± 2007.0 pg/mL), 89-fold higher on D9 (930.3 ± 306.4 pg/mL), and 3-fold higher on D176 32.2 ± 8.9 pg/mL after TBI compared with baseline (10.5 ± 2.6 pg/mL; all p < 0.001). Plasma NF-L levels distinguished TBI rats from naïve animals at all time-points examined (area under the curve [AUC] 1.0, p < 0.001), and from sham-operated controls on D2 (AUC 1.0, p < 0.001). Plasma NF-L increases on D2 were associated with somatomotor impairment severity (ρ = −0.480, p < 0.05) and the cortical lesion extent in MRI (ρ = 0.401, p < 0.05). Plasma NF-L increases on D2 or D9 were associated with the cortical lesion extent in histologic sections at 6 months post-injury (ρ = 0.437 for D2; ρ = 0.393 for D9, p < 0.05). Plasma NF-L levels, however, did not predict somatomotor recovery, cognitive decline, or epileptogenesis (p > 0.05). Plasma NF-L levels represent a promising noninvasive translational diagnostic biomarker for acute TBI and a prognostic biomarker for post-injury somatomotor impairment and long-term structural brain damage.

Noninvasive, affordable circulating biomarkers for difficult-to-diagnose mild traumatic brain injury (mTBI) are an unmet medical need. Although blood microRNA (miRNA) levels are reportedly altered after traumatic brain injury (TBI), their diagnostic potential for mTBI remains inconclusive. We hypothesized that acutely altered plasma miRNAs could serve as diagnostic biomarkers both in the lateral fluid percussion injury (FPI) model and clinical mTBI. We performed plasma small RNA-sequencing from adult male Sprague–Dawley rats (n = 31) at 2 days post-TBI, followed by polymerase chain reaction (PCR)-based validation of selected candidates. miR-9a-3p, miR-136-3p, and miR-434-3p were identified as the most promising candidates at 2 days after lateral FPI. Digital droplet PCR (ddPCR) revealed 4.2-, 2.8-, and 4.6-fold elevations in miR-9a-3p, miR-136-3p, and miR-434-3p levels (p < 0.01 for all), respectively, distinguishing rats with mTBI from naïve rats with 100% sensitivity and specificity. DdPCR further identified a subpopulation of mTBI patients with plasma miR-9-3p (n = 7/15) and miR-136-3p (n = 5/15) levels higher than one standard deviation above the control mean at <2 days postinjury. In sTBI patients, plasma miR-9-3p levels were 6.5- and 9.2-fold in comparison to the mTBI and control groups, respectively. Thus, plasma miR-9-3p and miR-136-3p were identified as promising biomarker candidates for mTBI requiring further evaluation in a larger patient population.

Traumatic brain injury (TBI) is associated with the pathological activation of immune‐competent cells in the brain, such as astrocytes, microglia and infiltrating immune blood cells, resulting in chronic inflammation and gliosis. This may contribute to the secondary injury after TBI, thus understanding of these processes is crucial for the development of effective treatments of post‐traumatic pathologies. MicroRNAs (miRNAs, miRs) are small noncoding RNAs, functioning as posttranscriptional regulators of gene expression. The increased expression of inflammation‐associated microRNAs miR155 and miR142 has been reported after TBI in rats. However, expression of these miRNAs in the human brain post‐TBI is not studied and their functions are not well understood. Moreover, circulating miR155 and miR142 are candidate biomarkers. Therefore, we characterized miR142 and miR155 expression in the perilesional cortex and plasma of rats that underwent lateral fluid‐percussion injury, a model for TBI and in the human perilesional cortex post‐TBI. We demonstrated higher miR155 and miR142 expression in the perilesional cortex of rats 2 weeks post‐TBI. In plasma, miR155 was associated with proteins and miR142 with extracellular vesicles, however their expression did not change. In the human perilesional cortex miR155 was most prominently expressed by activated astrocytes, whereas miR142 was expressed predominantly by microglia, macrophages and lymphocytes. Pro‐inflammatory medium from macrophage‐like cells stimulated miR155 expression in astrocytes and overexpression of miR142 in these cells further potentiated a pro‐inflammatory state of activated astrocytes. We conclude that miR155 and miR142 promote brain inflammation via astrocyte activation and may be involved in the secondary brain injury after TBI.

Extracellular vesicles (EVs) are small vesicles involved in intercellular communication. Data is emerging that EVs and their cargo have potential as diagnostic biomarkers and treatments for brain diseases, including traumatic brain injury and epilepsy. Here, we summarize the current knowledge regarding changes in EV numbers and cargo in status epilepticus (SE) and traumatic brain injury (TBI), which are clinically significant etiologies for acquired epileptogenesis in animals and humans. We also review encouraging data, which suggests that EVs secreted by stem cells may serve as recovery-enhancing treatments for SE and TBI. Using Gene Set Enrichment Analysis, we show that brain EV-related transcripts are positively enriched in rodent models of epileptogenesis and epilepsy, and altered in response to anti-seizure drugs. These data suggest that EVs show promise as biomarkers, treatments and drug targets for epilepsy. In parallel to gathering conceptual knowledge, analytics platforms for the isolation and analysis of EV contents need to be further developed.

The microRNA (miRNA) cargo contained in plasma extracellular vesicles (EVs) offers a relatively little explored source of biomarkers for brain diseases that can be obtained noninvasively. Methods to isolate EVs from plasma, however, are still being developed. For EV isolation, it is important to ensure the removal of vesicle‐free miRNAs, which account for approximately two‐thirds of plasma miRNAs. Membrane particle precipitation‐based EV isolation is an appealing method because of the simple protocol and high yield. Here, we evaluated the performance of a precipitation‐based method to obtain enriched EV‐specific miRNAs from a small volume of rat plasma. We performed size‐exclusion chromatography (SEC) on precipitation‐isolated EV pellets and whole plasma. The SEC fractions were analysed using Nanoparticle Tracking Analysis (NTA), protein and miRNA concentration assays, and droplet digital polymerase chain reaction for four miRNAs (miR‐142‐3p, miR‐124‐3p, miR‐23a, miR‐122). Precipitation‐isolated EVs and selected SEC fractions from the plasma were also analysed with transmission electron microscopy (TEM). Precipitation‐based EV isolation co‐precipitated 9% to 15% of plasma proteins and 21% to 99% of vesicle‐free miRNAs, depending on the individual miRNAs. In addition, the amount of miR‐142‐3p, found mainly in EV fractions, was decreased in the EV fractions, indicating that part of it was lost during precipitation‐based isolation. Western blot and TEM revealed both protein and lipoprotein contamination in the precipitation‐isolated EV‐pellets. Our findings indicate that a precipitation‐based method is not sufficient for purifying plasma EV‐contained miRNA cargo. The particle number measured by NTA is high, but this is mostly due to the contaminating lipoproteins. Although a part of the vesicle‐free miRNA is removed, vesicle‐free miRNA still dominates in plasma EV pellets isolated by the precipitation‐based method.