Development of Prolonged Focal Cerebral Edema and Regional Cation Changes Following Experimental Brain Injury in the Rat

The present study examined the formation of regional cerebral edema in adult rats subjected to lateral (parasagittal) experimental fluid-percussion brain injury. Animals receiving fluid-percussion brain injury of moderate severity over the left parietal cortex were assayed for brain water content at 6 h, 24 h, and 2, 3, 5, and 7 days post injury. Regional sodium and potassium concentrations were measured in a separate group of animals at 10 min, 1 h, 6 h, and 24 h following fluid-percussion injury. Injured parietal cortex demonstrated significant edema, beginning at 6 h post injury (p less than 0.05) and persisting up to 5 days post injury. In the hippocampus ipsilateral to the site of cortical injury, significant edema occurred as early as 1 h post injury (p less than 0.05), with resolution of water accumulation beginning at 3 days. Sodium concentrations significantly increased in both injured cortex (1 h post injury, p less than 0.05) and injured hippocampus (10 min post injury, p less than 0.05). Potassium concentrations fell significantly 1 h post injury within the injured cortex (p less than 0.05), whereas significant decreases were not observed until 24 h post injury within the injured hippocampus. Cation alterations persisted throughout the 24-h post injury period. These results demonstrate that regional brain edema and cation deregulation occur in rats subjected to lateral fluid-percussion brain injury and that these changes may persist for a prolonged period after brain injury.     

A Novel Model of Mild Traumatic Brain Injury for Juvenile Rats

Despite growing evidence that childhood represents a major risk period for mild traumatic brain injury (mTBI) from sports-related concussions, motor vehicle accidents, and falls, a reliable animal model of mTBI had previously not been developed for this important aspect of development. The modified weight-drop technique employs a glancing impact to the head of a freely moving rodent transmitting acceleration, deceleration, and rotational forces upon the brain. When applied to juvenile rats, this modified weight-drop technique induced clinically relevant behavioural outcomes that were representative of post-concussion symptomology. The technique is a rapidly applied procedure with an extremely low mortality rate, rendering it ideal for high-throughput studies of therapeutics. In addition, because the procedure involves a mild injury to a closed head, it can easily be used for studies of repetitive brain injury. Owing to the simplistic nature of this technique, and the clinically relevant biomechanics of the injury pathophysiology, the modified weight-drop technique provides researchers with a reliable model of mTBI that can be used in a wide variety of behavioural, molecular, and genetic studies.     

Significance of Ubiquitin Carboxy-Terminal Hydrolase L1 Elevations in Athletes after Sub-Concussive Head Hits

The impact of sub-concussive head hits (sub-CHIs) has been recently investigated in American football players, a population at risk for varying degrees of post-traumatic sequelae. Results show how sub-CHIs in athletes translate in serum as the appearance of reporters of blood-brain barrier disruption (BBBD), how the number and severity of sub-CHIs correlate with elevations of putative markers of brain injury is unknown. Serum brain injury markers such as UCH-L1 depend on BBBD. We investigated the effects of sub-CHIs in collegiate football players on markers of BBBD, markers of cerebrospinal fluid leakage (serum beta 2-transferrin) and markers of brain damage. Emergency room patients admitted for a clinically-diagnosed mild traumatic brain injury (mTBI) were used as positive controls. Healthy volunteers were used as negative controls. Specifically this study was designed to determine the use of UCH-L1 as an aid in the diagnosis of sub-concussive head injury in athletes. The extent and intensity of head impacts and serum values of S100B, UCH-L1, and beta-2 transferrin were measured pre- and post-game from 15 college football players who did not experience a concussion after a game. S100B was elevated in players experiencing the most sub-CHIs; UCH-L1 levels were also elevated but did not correlate with S100B or sub-CHIs. Beta-2 transferrin levels remained unchanged. No correlation between UCH-L1 levels and mTBI were measured in patients. Low levels of S100B were able to rule out mTBI and high S100B levels correlated with TBI severity. UCH-L1 did not display any interpretable change in football players or in individuals with mild TBI. The significance of UCH-L1 changes in sub-concussions or mTBI needs to be further elucidated.     

Traumatic Brain Injury Dysregulates MicroRNAs to Modulate Cell Signaling in Rat Hippocampus

Traumatic brain injury (TBI) is a common cause for cognitive and communication problems, but the molecular and cellular mechanisms are not well understood. Epigenetic modifications, such as microRNA (miRNA) dysregulation, may underlie altered gene expression in the brain, especially hippocampus that plays a major role in spatial learning and memory and is vulnerable to TBI. To advance our understanding of miRNA in pathophysiological processes of TBI, we carried out a time-course microarray analysis of microRNA expression profile in rat ipsilateral hippocampus and examined histological changes, apoptosis and synapse ultrastructure of hippocampus post moderate TBI. We found that 10 out of 156 reliably detected miRNAs were significantly and consistently altered from one hour to seven days after injury. Bioinformatic and gene ontology analyses revealed 107 putative target genes, as well as several biological processes that might be initiated by those dysregulated miRNAs. Among those differentially expressed microRNAs, miR-144, miR-153 and miR-340-5p were confirmed to be elevated at all five time points after TBI by quantitative RT-PCR. Western blots showed three of the predicated target proteins, calcium/calmodulin-dependent serine protein kinase (CASK), nuclear factor erythroid 2-related factor 2 (NRF2) and alpha-synuclein (SNCA), were concurrently down- regulated, suggesting that miR-144, miR-153 and miR-340-5p may play important roles collaboratively in the pathogenesis of TBI-induced cognitive and memory impairments. These microRNAs might serve as potential targets for progress assessment and intervention against TBI to mitigate secondary damage to the brain.     

Neuropsychological Outcome and Diffusion Tensor Imaging in Complicated versus Uncomplicated Mild Traumatic Brain Injury

This study examined whether intracranial neuroimaging abnormalities in those with mild traumatic brain injury (MTBI) (i.e., “complicated” MTBIs) are associated with worse subacute outcomes as measured by cognitive testing, symptom ratings, and/or diffusion tensor imaging (DTI). We hypothesized that (i) as a group, participants with complicated MTBIs would report greater symptoms and have worse neurocognitive outcomes than those with uncomplicated MTBI, and (ii) as a group, participants with complicated MTBIs would show more Diffusion Tensor Imaging (DTI) abnormalities. Participants were 62 adults with MTBIs (31 complicated and 31 uncomplicated) who completed neurocognitive testing, symptom ratings, and DTI on a 3T MRI scanner approximately 6-8 weeks post injury. There were no statistically significant differences between groups on symptom ratings or on a broad range of neuropsychological tests. When comparing the groups using tract-based spatial statistics for DTI, no significant difference was found for axial diffusivity or mean diffusivity. However, several brain regions demonstrated increased radial diffusivity (purported to measure myelin integrity), and decreased fractional anisotropy in the complicated group compared with the uncomplicated group. Finally, when we extended the DTI analysis, using a multivariate atlas based approach, to 32 orthopedic trauma controls (TC), the findings did not reveal significantly more areas of abnormal DTI signal in the complicated vs. uncomplicated groups, although both MTBI groups had a greater number of areas with increased radial diffusivity compared with the trauma controls. This study illustrates that macrostructural neuroimaging changes following MTBI are associated with measurable changes in DTI signal. Of note, however, the division of MTBI into complicated and uncomplicated subtypes did not predict worse clinical outcome at 6-8 weeks post injury.     

高糖诱导内皮细胞损伤microRNA表达紊乱的体外研究

目的:研究高糖诱导的内皮细胞损伤微小RNA(microRNA,miRNA)的表达变化。方法:常规培养的人冠状动脉内皮细胞,利用不同浓度D-葡萄糖溶液(0 mmol/L、5 mmol/L、15 mmol/L和25 mmol/L),诱导刺激24 h后分别用CCK-8法和流式细胞术检测其生长活力和凋亡水平。收集细胞总RNA,利用实时定量PCR(Quantitative real-time PCR,q RT-PCR)检测miRNA的表达变化,同时利用TargetScan、PicTar等生物信息学预测软件预测可能的靶基因。结果:高糖溶液(25 mmol/L)刺激内皮细胞后,细胞生长活力明显降低,为对照组的67.5%(P0.01),凋亡水平为对照组的4.5倍(P0.01)。QRT-PCR结果显示miRNA的表达出现了明显的紊乱,其中miR-451、miR-504、miR-302d、miR-18b*、miR-198、miR-328和miR-517c明显下调,miR-29c、miR-100*、miR-137、miR-660和miR-217明显上调(P0.05)。靶基因预测发现miR-217和miR-451可能调控内皮细胞功能相关的多个基因的表达。结论:在高糖诱导的内皮细胞损伤中,miRNA表达紊乱提示其可能参与内皮细胞功能。     

Efficacy of N-Acetyl Cysteine in Traumatic Brain Injury

In this study, using two different injury models in two different species, we found that early post-injury treatment with N-Acetyl Cysteine (NAC) reversed the behavioral deficits associated with the TBI. These data suggest generalization of a protocol similar to our recent clinical trial with NAC in blast-induced mTBI in a battlefield setting [1], to mild concussion from blunt trauma. This study used both weight drop in mice and fluid percussion injury in rats. These were chosen to simulate either mild or moderate traumatic brain injury (TBI). For mice, we used novel object recognition and the Y maze. For rats, we used the Morris water maze. NAC was administered beginning 30–60 minutes after injury. Behavioral deficits due to injury in both species were significantly reversed by NAC treatment. We thus conclude NAC produces significant behavioral recovery after injury. Future preclinical studies are needed to define the mechanism of action, perhaps leading to more effective therapies in man.     

Regional Levels of Lactate and Norepinephrine After Experimental Brain Injury

Abstract: The recently developed controlled cortical impact model of brain injury in rats may be an excellent tool by which to attempt to understand the neurochemical mechanisms mediating the pathophysiology of traumatic brain injury. In this study, rats were subjected to lateral controlled cortical impact brain injury of low grade severity; their brains were frozen in situ at various times after injury to measure regional levels of lactate, high energy phosphates, and norepinephrine. Tissue lactate concentration in the injury site left cortex was increased in injured animals by sixfold at 30 min and twofold at 2.5 h and 24 h after injury ( p < 0.05). At all postinjury times, lactate concentration was also increased in injured animals by about twofold in the cortex and hippocampus adjacent to the injury site ( p < 0.05). No significant changes occurred in the levels of ATP and phosphocreatine in most of the brain regions of injured animals. However, in the primary site of injury (left cortex), phosphocreatine concentration was decreased by 40% in injured animals at 30 min after injury ( p < 0.05). The norepinephrine concentration was decreased in the injury site left cortex of injured animals by 38% at 30 min, 29% at 2.5 h, and 30% at 24 h after injury ( p < 0.05). The level of norepinephrine was also reduced by ∼20% in the cortex adjacent to the injury site in injured animals. The present results suggest that controlled cortical impact brain injury produces disorder in the neuronal oxidative and norepinephrine metabolism.     

Are mild head injuries as mild as we think? Neurobehavioral concomitants of chronic post-concussion syndrome

Background  

Mild traumatic brain injury (MTBI) can sometimes lead to persistent postconcussion symptoms. One well accepted hypothesis claims that chronic PCS has a neural origin, and is related to neurobehavioral deficits. But the evidence is not conclusive. In the attempt to characterise chronic MTBI consequences, the present experiment used a group comparison design, which contrasted persons (a) with MTBI and PCS, (b) MTBI without PCS, and (c) matched controls. We predicted that participants who have experienced MTBI but show no signs of PCS would perform similar to controls. At the same time, a subgroup of MTBI participants would show PCS symptoms and only these volunteers would have poorer cognitive performance. Thereby, the performance deficits should be most noticeable in participants with highest PCS severity.     

Long-term, efficient inhibition of microRNA function in mice using rAAV vectors

Understanding the function of individual microRNA (miRNA) species in mice would require the production of hundreds of loss-of-function strains. To accelerate analysis of miRNA biology in mammals, we combined recombinant adeno-associated virus (rAAV) vectors with miRNA 'tough decoys' (TuDs) to inhibit specific miRNAs. Intravenous injection of rAAV9 expressing anti-miR-122 or anti-let-7 TuDs depleted the corresponding miRNA and increased its mRNA targets. rAAV producing anti-miR-122 TuD but not anti-let-7 TuD reduced serum cholesterol by >30% for 25 weeks in wild-type mice. High-throughput sequencing of liver miRNAs from the treated mice confirmed that the targeted miRNAs were depleted and revealed that TuDs induced miRNA tailing and trimming in vivo. rAAV-mediated miRNA inhibition thus provides a simple way to study miRNA function in adult mammals and a potential therapy for dyslipidemia and other diseases caused by miRNA deregulation.     

Cognitive Impairments Accompanying Rodent Mild Traumatic Brain Injury Involve p53-Dependent Neuronal Cell Death and Are Ameliorated by the Tetrahydrobenzothiazole PFT-α

With parallels to concussive mild traumatic brain injury (mTBI) occurring in humans, anesthetized mice subjected to a single 30 g weight drop mTBI event to the right parietal cortex exhibited significant diffuse neuronal degeneration that was accompanied by delayed impairments in recognition and spatial memory. To elucidate the involvement of reversible p53-dependent apoptosis in this neuronal loss and associated cognitive deficits, mice were subjected to experimental mTBI followed by the systemic administration of the tetrahydrobenzothiazole p53 inactivator, PFT-α, or vehicle. Neuronal loss was quantified immunohistochemically at 72 hr. post-injury by the use of fluoro-Jade B and NeuN within the dentate gyrus on both sides of the brain, and recognition and spatial memory were assessed by novel object recognition and Y-maze paradigms at 7 and 30 days post injury. Systemic administration of a single dose of PFT-α 1 hr. post-injury significantly ameliorated both neuronal cell death and cognitive impairments, which were no different from sham control animals. Cellular studies on human SH-SY5Y cells and rat primary neurons challenged with glutamate excitotoxicity and H₂O₂ induced oxidative stress, confirmed the ability of PFT-α and a close analog to protect against these TBI associated mechanisms mediating neuronal loss. These studies suggest that p53-dependent apoptotic mechanisms underpin the neuronal and cognitive losses accompanying mTBI, and that these are potentially reversible by p53 inactivation.     

FK506 Protects Neurons Following Peripheral Nerve Injury Via Immunosuppression

In this study, we have evaluated neuroprotective effect of an immunosuppressant immunophilin ligand, FK506, in the sciatic nerve injury model in rats. FK506 was injected to the sciatic nerve transected 3-month-old female Wistar rats (2 mg/kg/day starting 1 day prior to sciatic nerve injury up to 7 day post operation). Equal number of sciatic nerve transected animals served as injured untreated controls. The contralateral side served as respective control. L4-L5 region of the spinal cord was removed on day 1, 3, 7, 14, 21, and 28, post operation and then processed for cryo-sectioning and paraffin sectioning. The cryocut sections were used for immunohistochemistry for localizing all microglia (using anti-Iba-1) and MHC-II expressing microglia (with OX-6). The physical dissector method was applied on Nissl stained paraffin sections for absolute motor neuron counting in the L4-L5 region of spinal cord. FK506 treated animals presented 88.7% neuronal survival while the injured alone had 79.12%, which is significantly less than the treated animals. FK506 caused early proliferation of microglia at 1 and 3 days post operation. FK506 also significantly restricted transformation of these cells in to phagocytes. Colocalization of activated microglia by anti-Iba-1 and OX-6 antibodies, confirms that the MHC-II expressing cells in injured spinal cord are none other than microglial cells and MHC-II expressing cells are significantly less in treated as compared to untreated injured animals. We propose that immunosuppression is one of the main mechanisms by which FK506 protects the central neurons following peripheral injury.     

Nerve Growth Factor Administration Attenuates Cognitive but Not Neurobehavioral Motor Dysfunction or Hippocampal Cell Loss Following Fluid-Percussion Brain Injury in Rats

Abstract: Lateral fluid-percussion brain injury in rats results in cognitive deficits, motor dysfunction, and selective hippocampal cell loss. Neurotrophic factors have been shown to have potential therapeutic applications in neurodegenerative diseases, and nerve growth factor (NGF) has been shown to be neuroprotective in models of excitotoxicity. This study evaluated the neuroprotective efficacy of intracerebral NGF infusion after traumatic brain injury. Male Sprague-Dawley rats received lateral fluid-percussion brain injury of moderate severity (2.1–2.3 atm). A miniosmotic pump was implanted 24 h after injury to infuse NGF (n = 34) or vehicle (n = 16) directly into the region of maximal cortical injury. Infusions of NGF continued until the animal was killed at 72 h, 1 week, or 2 weeks after injury. Animals were evaluated for cognitive dysfunction (Morris Water Maze) and regional neuronal cell loss (Nissl staining) at each of the three time points. Animals surviving for 1 or 2 weeks were also evaluated for neurobehavioral motor function. Although an improvement in memory scores was not observed at 72 h after injury, animals receiving NGF infusions showed significantly improved memory scores when tested at 1 or 2 weeks after injury compared with injured animals receiving vehicle infusions ( p < 0.05). Motor scores and CA3 hippocampal cell loss were not significantly different in any group of NGF-treated animals when compared with controls. These data suggest that NGF administration, in the acute, posttraumatic period following fluid-percussion brain injury, may have potential in improving post-traumatic cognitive deficits.     

Voluntary Alcohol Intake following Blast Exposure in a Rat Model of Mild Traumatic Brain Injury

Alcoholism is a frequent comorbidity following mild traumatic brain injury (mTBI), even in patients without a previous history of alcohol dependence. Despite this correlational relationship, the extent to which the neurological effects of mTBI contribute to the development of alcoholism is unknown. In this study, we used a rodent blast exposure model to investigate the relationship between mTBI and voluntary alcohol drinking in alcohol naïve rats. We have previously demonstrated in Sprague Dawley rats that blast exposure leads to microstructural abnormalities in the medial prefrontal cortex (mPFC) and other brain regions that progress from four to thirty days. The mPFC is a brain region implicated in alcoholism and drug addiction, although the impact of mTBI on drug reward and addiction using controlled models remains largely unexplored. Alcohol naïve Sprague Dawley rats were subjected to a blast model of mTBI (or sham conditions) and then tested in several common measures of voluntary alcohol intake. In a seven-week intermittent two-bottle choice alcohol drinking test, sham and blast exposed rats had comparable levels of alcohol intake. In a short access test session at the conclusion of the two-bottle test, blast rats fell into a bimodal distribution, and among high intake rats, blast treated animals had significantly elevated intake compared to shams. We found no effect of blast when rats were tested for an alcohol deprivation effect or compulsive drinking in a quinine adulteration test. Throughout the experiment, alcohol drinking was modest in both groups, consistent with other studies using Sprague Dawley rats. In conclusion, blast exposure had a minimal impact on overall alcohol intake in Sprague Dawley rats, although intake was increased in a subpopulation of blast animals in a short access session following intermittent access exposure.     

MicroRNA and mRNA Signatures in Ischemia Reperfusion Injury in Heart Transplantation

Ischemia reperfusion (I/R) injury is an unavoidable event occurring during heart transplantation, leading to graft failures and lower long-term survival rate of the recipient. Several studies have demonstrated that microRNAs (miRNAs) are vital regulators of signalling pathways involved in I/R injury. The present study aims to quantify the altered expression levels of miRNA and mRNA upon I/R injury in a mouse heart transplantation model, and to investigate whether these miRNA can regulate genes involved in I/R injury. We performed heterotopic heart transplantation on mouse models to generate heart tissue samples with I/R and non-I/R (control). The expression levels of miRNAs as well as genes were measured in heart grafts by microarray and real time RT-PCR. miRNA alteration in cardiomyocytes exposed to hypoxia was also detected by qRT-PCR. We observed significant alterations in miRNA and gene expression profile after I/R injury. There were 39 miRNAs significantly downregulated and 20 upregulated up to 1.5 fold in heart grafts with I/R injury compared with the grafts without I/R. 48 genes were observed with 3 fold change and p<0.05 and 18 signalling pathways were enriched using Keggs pathway library. Additionally, hypoxia/reperfusion induced primary cardiomyocyte apoptosis and altered miRNA expression profiles. In conclusion, this is the first report on miRNA expression profile for heart transplantation associated with I/R injury. These findings provide us with an insight into the role of miRNA in I/R injury in heart transplantation.     

Quantitative analysis of brain microstructure following mild blunt and blast trauma

We induced mild blunt and blast injuries in rats using a custom-built device and utilized in-house diffusion tensor imaging (DTI) software to reconstruct 3-D fiber tracts in brains before and after injury (1, 4, and 7 days). DTI measures such as fiber count, fiber length, and fractional anisotropy (FA) were selected to characterize axonal integrity. In-house image analysis software also showed changes in parameters including the area fraction (AF) and nearest neighbor distance (NND), which corresponded to variations in the microstructure of Hematoxylin and Eosin (H&E) brain sections. Both blunt and blast injuries produced lower fiber counts, but neither injury case significantly changed the fiber length. Compared to controls, blunt injury produced a lower FA, which may correspond to an early onset of diffuse axonal injury (DAI). However, blast injury generated a higher FA compared to controls. This increase in FA has been linked previously to various phenomena including edema, neuroplasticity, and even recovery. Subsequent image analysis revealed that both blunt and blast injuries produced a significantly higher AF and significantly lower NND, which correlated to voids formed by the reduced fluid retention within injured axons. In conclusion, DTI can detect subtle pathophysiological changes in axonal fiber structure after mild blunt and blast trauma. Our injury model and DTI method provide a practical basis for studying mild traumatic brain injury (mTBI) in a controllable manner and for tracking injury progression. Knowledge gained from our approach could lead to enhanced mTBI diagnoses, biofidelic constitutive brain models, and specialized pharmaceutical treatments.     

Increased Gray Matter Diffusion Anisotropy in Patients with Persistent Post-Concussive Symptoms following Mild Traumatic Brain Injury

A significant percentage of individuals diagnosed with mild traumatic brain injury (mTBI) experience persistent post-concussive symptoms (PPCS). Little is known about the pathology of these symptoms and there is often no radiological evidence based on conventional clinical imaging. We aimed to utilize methods to evaluate microstructural tissue changes and to determine whether or not a link with PPCS was present. A novel analysis method was developed to identify abnormalities in high-resolution diffusion tensor imaging (DTI) when the location of brain injury is heterogeneous across subjects. A normative atlas with 145 brain regions of interest (ROI) was built from 47 normal controls. Comparing each subject’s diffusion measures to the atlas generated subject-specific profiles of injury. Abnormal ROIs were defined by absolute z-score values above a given threshold. The method was applied to 11 PPCS patients following mTBI and 11 matched controls. Z-score information for each individual was summarized with two location-independent measures: “load” (number of abnormal regions) and “severity” (largest absolute z-score). Group differences were then computed using Wilcoxon rank sum tests. Results showed statistically significantly higher load (p = 0.018) and severity (p = 0.006) for fractional anisotropy (FA) in patients compared with controls. Subject-specific profiles of injury evinced abnormally high FA regions in gray matter (30 occurrences over 11 patients), and abnormally low FA in white matter (3 occurrences over 11 subjects). Subject-specific profiles provide important information regarding the pathology associated with PPCS. Increased gray matter (GM) anisotropy is a novel in-vivo finding, which is consistent with an animal model of brain trauma that associates increased FA in GM with pathologies such as gliosis. In addition, the individualized analysis shows promise for enhancing the clinical care of PPCS patients as it could play a role in the diagnosis of brain injury not revealed using conventional imaging.     

A set of microRNAs mediate direct conversion of human umbilical cord lining-derived mesenchymal stem cells into hepatocytes

In a previous study, we elucidated the specific microRNA (miRNA) profile of hepatic differentiation. In this study, we aimed to clarify the instructive role of six overexpressed miRNAs (miR-1246, miR-1290, miR-148a, miR-30a, miR-424 and miR-542-5p) during hepatic differentiation of human umbilical cord lining-derived mesenchymal stem cells (hMSCs) and to test whether overexpression of any of these miRNAs is sufficient to induce differentiation of the hMSCs into hepatocyte-like cells. Before hepatic differentiation, hMSCs were infected with a lentivirus containing a miRNA inhibitor sequence. We found that downregulation of any one of the six hepatic differentiation-specific miRNAs can inhibit HGF-induced hepatic differentiation including albumin expression and LDL uptake. Although overexpression of any one of the six miRNAs alone or liver-enriched miR-122 cannot initiate hepatic differentiation, ectopic overexpression of seven miRNAs (miR-1246, miR-1290, miR-148a, miR-30a, miR-424, miR-542-5p and miR-122) together can stimulate hMSC conversion into functionally mature induced hepatocytes (iHep). Additionally, after transplantation of the iHep cells into mice with CCL4-induced liver injury, we found that iHep not only can improve liver function but it also can restore injured livers. The findings from this study indicate that miRNAs have the capability of directly converting hMSCs to a hepatocyte phenotype in vitro.     

Integrated analyses of zebrafish miRNA and mRNA expression profiles identify miR-29b and miR-223 as potential regulators of optic nerve regeneration

Background

Unlike mammals, zebrafish have the ability to regenerate damaged parts of their central nervous system (CNS) and regain functionality of the affected area. A better understanding of the molecular mechanisms involved in zebrafish regeneration may therefore provide insight into how CNS repair might be induced in mammals. Although many studies have described differences in gene expression in zebrafish during CNS regeneration, the regulatory mechanisms underpinning the differential expression of these genes have not been examined.

Results

We used microarrays to analyse and integrate the mRNA and microRNA (miRNA) expression profiles of zebrafish retina after optic nerve crush to identify potential regulatory mechanisms that underpin central nerve regeneration. Bioinformatic analysis identified 3 miRNAs and 657 mRNAs that were differentially expressed after injury. We then combined inverse correlations between our miRNA expression and mRNA expression, and integrated these findings with target predictions from TargetScan Fish to identify putative miRNA-gene target pairs. We focused on two over-expressed miRNAs (miR-29b and miR-223), and functionally validated seven of their predicted gene targets using RT-qPCR and luciferase assays to confirm miRNA-mRNA binding. Gene ontology analysis placed the miRNA-regulated genes (eva1a, layna, nefmb, ina, si:ch211-51a6.2, smoc1, sb:cb252) in key biological processes that included cell survival/apoptosis, ECM-cytoskeleton signaling, and heparan sulfate proteoglycan binding,

Conclusion

Our results suggest a key role for miR-29b and miR-223 in zebrafish regeneration. The identification of miRNA regulation in a zebrafish injury model provides a framework for future studies in which to investigate not only the cellular processes required for CNS regeneration, but also how these mechanisms might be regulated to promote successful repair and return of function in the injured mammalian brain.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1772-1) contains supplementary material, which is available to authorized users.