Controlled Cortical Impact Group Research Articles

The Role of the Orexin System in Craniocerebral Trauma-Induced Epilepsy in Mice.

Following traumatic brain injury (TBI), an imbalance arises in the central nervous system within the hippocampus region, resulting in the proliferation of mossy cell fibers, causing abnormal membrane discharge. Moreover, disruptions in cellular neurotransmitter secretion induce post-traumatic epilepsy. Extensive experimental and clinical data indicate that the orexin system plays a regulatory role in the hippocampal central nervous system, but the specific regulatory effects are unclear. Therefore, further experimental evaluation of its relevance is needed. This study aims to investigate the effects of orexin receptor agonists (OXA) on the seizure threshold and intensity in controlled cortical impact (CCI) mice, and to understand the role of the orexin system in post-traumatic epilepsy (PTE). Male C57BL/6 mice weighing 18-22 g were randomly divided into three groups: Sham, CCI, and CCI+OXA. The three groups of mice were sequentially constructed with models, implanted with electrodes, and established drug-delivery cannulas. After a 30-day recovery, the Sham and CCI groups were injected with physiological saline through the administration cannulas, while the CCI+OXA group was injected with OXA. Subsequently, all mice underwent electrical stimulation every 30 minutes for a total of 15 times. Epileptic susceptibility, duration, intensity, and cognitive changes were observed. Concurrently, the expression levels and changes of GABAergic neurons in the hippocampus of each group were examined by immunofluorescence. Injecting OXA into hippocampal CA1 reduces the threshold of post-traumatic seizures, prolongs the post-discharge duration, prolongs seizure duration, reduces cognitive ability, and exacerbates the loss of GABAergic neurons in the hippocampal region. Based on the results, we can find that injecting OXA antagonists into the CA1 region of the hippocampus can treat or prevent the occurrence and progression of post-traumatic epilepsy.

Chitosan revokes controlled-cortical impact generated neurological aberrations in circadian disrupted mice via TLR4-NLRP3 axis

The severity of inevitable neurological deficits and long-term psychiatric disorders in the aftermath of traumatic brain injury is influenced by pre-injury biological factors. Herein, we investigated the therapeutic effect of chitosan lactate on neurological and psychiatric aberrations inflicted by circadian disruption (CD) and controlled-cortical impact (CCI) injury in mice. Firstly, CD was developed in mice by altering sporadic day-night cycles for 2 weeks. Then, CCI surgery was performed using a stereotaxic ImpactOne device. Mice subjected to CCI displayed a significant disruption of motor coordination at 1-, 3- and 5-days post-injury (DPI) in the rotarod test. These animals showed anxiety- and depression-like behaviors in the elevated plus maze and forced-swim test at 14 and 15 DPI, respectively. Notably, mice subjected to CD + CCI exhibited severe cognitive impairment in Y-maze and novel object recognition tasks. The compromised neurological, psychiatric, and cognitive functions were mitigated in chitosan-treated mice (1 and 3 mg/mL). Immunohistochemistry and real-time PCR assay results revealed the magnified responses of prima facie biomarkers like glial-fibrillary acidic protein and ionized calcium-binding adaptor molecule 1 in the pericontusional brain region of the CD + CCI group, indicating aggravated inflammation. We also noted the depleted levels of brain-derived neurotrophic factor and augmented expression of toll-like receptor 4 (TLR4)-leucine-rich-containing family pyrin domain-containing 3 (NLRP3) signaling [apoptosis-associated-speck-like protein (ASC), caspase-1, and interleukin 1-β] in the pericontusional area of CD + CCI group. CCI-induced changes in the astrocyte-glia and aggravated immune responses were ameliorated in chitosan-treated mice. These results suggest that the neuroprotective effect of chitosan in CCI-induced brain injury may be mediated by inhibition of the TLR4-NLRP3 axis.

Long-term effects of myo-inositol on traumatic brain injury: Epigenomic and transcriptomic studies

Background and purposeTraumatic brain injury (TBI) and its consequences remain great challenges for neurology. Consequences of TBI are associated with various alterations in the brain but little is known about long-term changes of epigenetic DNA methylation patterns. Moreover, nothing is known about potential treatments that can alter these epigenetic changes in beneficial ways. Therefore, we have examined myo-inositol (MI), which has positive effects on several pathological conditions. MethodsTBI was induced in mice by controlled cortical impact (CCI). One group of CCI animals received saline injections for two months (TBI+SAL), another CCI group received MI treatment (TBI+MI) for the same period and one group served as a sham-operated control. Mice were sacrificed 4 months after CCI and changes in DNA methylome and transcriptomes were examined. ResultsFor the first time we: (i) provide comprehensive map of long-term DNA methylation changes after CCI in the hippocampus; (ii) identify differences by methylation sites between the groups; (iii) characterize transcriptome changes; (iv) provide association between DNA methylation sites and gene expression. MI treatment is linked with upregulation of genes covering 33 biological processes, involved in immune response and inflammation. In support of these findings, we have shown that expression of BATF2, a transcription factor involved in immune-regulatory networks, is upregulated in the hippocampus of the TBI+MI group where the BATF2 gene is demethylated. ConclusionTBI is followed by long-term epigenetic and transcriptomic changes in hippocampus. MI treatment has a significant effect on these processes by modulation of immune response and biological pathways of inflammation.

Hyperbaric oxygen therapy alleviates intestinal dysfunction following traumatic brain injury via m6A regulation.

Hyperbaric oxygen (HBO) therapy can attenuate neurological impairment after traumatic brain injury (TBI) and alleviate intestinal dysfunction. However, the role and mechanism of HBO therapy in intestinal dysfunction following TBI remain unclear. Herein, by establishing a mouse model of controlled cortical impact (CCI), we found that HBO therapy reduced histopathological lesions and decreased the levels of inflammatory and oedema proteins in the intestinal tissues of mice 10 days after TBI. We also showed that HBO therapy improved microbiome abundance and probiotic (particularly g_Bifidobacterium) colonisation in mice post-CCI. Then, we identified that the m6A level imcreased notably in injured cortical tissue of CCI+HBO group compared with the CCI group following CCI. Thus, our results suggested that HBO therapy could alleviate TBI-induced intestinal dysfunction and m6A might participate in this regulation process, which provides new insights for exploring the specific mechanism and targets of HBO in the treatment of intestinal dysfunction after TBI, thereby improving the therapeutic effect of HBO.

Acute brain injury increases pulmonary capillary permeability via sympathetic activation-mediated high fluid shear stress and destruction of the endothelial glycocalyx layer

Neurogenic pulmonary edema secondary to acute brain injury (ABI) is a common and fatal disease condition. However, the pathophysiology of brain-lung interactions is incompletely understood. This study aims to investigate whether sympathetic activation-mediated high fluid shear stress after ABI would damage pulmonary endothelial glycocalyx thus leading to increased pulmonary capillary permeability. The tricuspid annular plane systolic excursion (TAPSE) was detected in a rat model of controlled cortical impact (CCI) and CCI + transection of the cervical sympathetic trunk (TCST). Changes in pulmonary capillary permeability were assessed by analyzing the Evans blue, measuring the dry/wet weight ratio of the lungs and altering protein levels in the bronchoalveolar lavage fluid (BALF). The parallel-plate flow chamber system was used to simulate the fluid shear stress in vitro. Western blotting and immunofluorescence staining were used to determine the expression levels of hyaluronan-binding protein (CEMIP), syndecan-1 and tight junction proteins (TJPs, including claudin-5 and occludin). TCST could restrain cardiac overdrive and sympathetic activation in a rat model of CCI. Compared to the CCI group, the CCI + TCST group showed a reduction of CEMPI (which degrades hyaluronic acid), along with an increase of syndecan-1 and TJPs. CCI + TCST group presented decreasing pulmonary capillary permeability. In vitro, high shear stress (HSS) increased the expression of CEMIP and reduced syndecan-1 and TJPs, which was coordinated with the results in vivo. Our findings show that sympathetic activation-mediated high fluid shear stress after ABI would damage pulmonary endothelial glycocalyx thus leading to increased pulmonary capillary permeability.

Hyperbaric Oxygen Therapy Alleviates Memory and Motor Impairments Following Traumatic Brain Injury via the Modulation of Mitochondrial-Dysfunction-Induced Neuronal Apoptosis in Rats.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality in young adults, characterized by primary and secondary injury. Primary injury is the immediate mechanical damage, while secondary injury results from delayed neuronal death, often linked to mitochondrial damage accumulation. Hyperbaric oxygen therapy (HBOT) has been proposed as a potential treatment for modulating secondary post-traumatic neuronal death. However, the specific molecular mechanism by which HBOT modulates secondary brain damage through mitochondrial protection remains unclear. Spatial learning, reference memory, and motor performance were measured in rats before and after Controlled Cortical Impact (CCI) injury. The HBOT (2.5 ATA) was performed 4 h following the CCI and twice daily (12 h intervals) for four consecutive days. Mitochondrial functions were assessed via high-resolution respirometry on day 5 following CCI. Moreover, IHC was performed at the end of the experiment to evaluate cortical apoptosis, neuronal survival, and glial activation. The current result indicates that HBOT exhibits a multi-level neuroprotective effect. Thus, we found that HBOT prevents cortical neuronal loss, reduces the apoptosis marker (cleaved-Caspase3), and modulates glial cell proliferation. Furthermore, HBO treatment prevents the reduction in mitochondrial respiration, including non-phosphorylation state, oxidative phosphorylation, and electron transfer capacity. Additionally, a superior motor and spatial learning performance level was observed in the CCI group treated with HBO compared to the CCI group. In conclusion, our findings demonstrate that HBOT during the critical period following the TBI improves cognitive and motor damage via regulating glial proliferation apoptosis and protecting mitochondrial function, consequently preventing cortex neuronal loss.

Chronic juvenile stress exacerbates neurobehavioral dysfunction and neuroinflammation following traumatic brain injury in adult mice.

Chronic stress in adolescence may affect brain maturation and predispose individuals to psychiatric disorders in adulthood. However, whether chronic juvenile stress influences vulnerability to nonpsychiatric brain injuries, such as traumatic brain injury (TBI), remains unclear. Therefore, we hypothesized that juvenile stress-related neuronal circuit disturbances could aggravate brain damage following TBI in adulthood. For chronic stress, we used an unpredictable chronic mild stress (UCMS) procedure for 5 weeks in adolescent mice. This was followed by a controlled cortical impact (CCI) injury to evaluate the influence of chronic juvenile stress on brain damage progression following TBI in adult mice. Mice underwent UCMS alone, UCMS followed by CCI, CCI alone, or sham operation. We characterized neurobehavioral deficits (Barnes maze, open field, and light-dark tests), neuroinflammation (ionized calcium-binding adapter molecule 1 [Iba-1], glial fibrillary acidic protein [GFAP], and neuron-specific nuclear protein [NeuN] immunoreactivity), and apoptosis (B-cell lymp [Bcl-2], Bcl-2-associated X protein [Bax], and procaspase-3 immunoreactivity). Following CCI, mice exposed to UCMS showed decreased spatial learning and memory in the Barnes maze test compared with unstressed mice. A significant increase in Iba-1, GFAP, and Bax/Bcl-2 immunostaining levels was observed in the mice exposed to UCMS followed by CCI compared with the CCI-only mice. In contrast, a significant decrease in NeuN immunostaining levels was observed in the UCMS with CCI group compared with the CCI alone group. Chronic stress in a juvenile mouse model aggravates neurobehavioral impairments and potentiates glial reactivity, neuronal injury, and apoptosis following moderate-to-severe TBI that occurs in adulthood. The present study suggests that juvenile chronic stress may influence poor outcomes following TBI in later adulthood.

Neuroblasts migration under control of reactive astrocyte-derived BDNF: a promising therapy in late neurogenesis after traumatic brain injury

BackgroundTraumatic brain injury (TBI) is a disease with high mortality and morbidity, which leads to severe neurological dysfunction. Neurogenesis has provided therapeutic options for treating TBI. Brain derived neurotrophic factor (BDNF) plays a key role in neuroblasts migration. We aimed to investigate to the key regulating principle of BDNF in endogenous neuroblasts migration in a mouse TBI model.MethodsIn this study, controlled cortical impact (CCI) mice (C57BL/6J) model was established to mimic TBI. The sham mice served as control. Immunofluorescence staining and enzyme-linked immunosorbent assay were performed on the CCI groups (day 1, 3, 7, 14 and 21 after CCI) and the sham group. All the data were analyzed with Student’s t-test or one-way or two-way analysis of variance followed by Tukey’s post hoc test.ResultsOur results revealed that neuroblasts migration initiated as early as day 1, peaking at day 7, and persisted till day 21. The spatiotemporal profile of BDNF expression was similar to that of neuroblasts migration, and BDNF level following CCI was consistently higher in injured cortex than in subventricular zone (SVZ). Reactive astrocytes account for the major resource of BDNF along the migrating path, localized with neuroblasts in proximity. Moreover, injection of exogenous CC chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1, at random sites promoted neuroblasts migration and astrocytic BDNF expression in both normal and CCI mice (day 28). These provoked neuroblasts can also differentiate into mature neurons. CC chemokine ligand receptor 2 antagonist can restrain the neuroblasts migration after TBI.ConclusionsNeuroblasts migrated along the activated astrocytic tunnel, directed by BDNF gradient between SVZ and injured cortex after TBI. CCL2 might be a key regulator in the above endogenous neuroblasts migration. Moreover, delayed CCL2 administration may provide a promising therapeutic strategy for late neurogenesis post-trauma.

Dual orexin antagonist normalized sleep homeostatic drive, enhanced GABAergic inhibition, and suppressed seizures after traumatic brain injury.

Traumatic brain injury (TBI) can result in posttraumatic epilepsy (PTE) and sleep disturbances. We hypothesized that treatment with sleep aids after TBI can ameliorate PTE. CD-1 mice underwent controlled cortical impact (CCI), sham injury, or no craniotomy. Sham and CCI groups underwent a monthlong daily treatment with sleep aids including a dual orexin antagonist (DORA-22) or THIP (gaboxadol) or a respective vehicle starting on the day of CCI. We performed continuous EEG (electroencephalography) recordings at week 1 and months 1, 2, and 3 for ~1 week each time. Seizure analysis occurred at all-time points and sleep analysis occurred in week 1 and month-1/2 in all groups. Subsets of CCI and sham groups were subjected to voltageclamp experiments in hippocampal slices to evaluate GABAergic synaptic inhibition. DORA-22 treatment suppressed seizures in month 1-3 recordings. TBI reduced the amplitude and frequency of miniature inhibitory synaptic currents (mIPSCs) in dentate granule cells and these changes were rescued by DORA-22 treatment. Sleep analysis showed that DORA-22 increased nonrapid eye movement (NREM) sleep during lights-off whereas THIP increased REM sleep during lights-on in week 1. Both treatments displayed subtle changes in time spent in NREM or REM at month-1/2 as well. TBI not only increased normalized EEG delta power (NΔ) at week-1 and month-1 but also resulted in the loss of the homeostatic diurnal oscillation of NΔ, which was restored by DORA-22 but not THIP treatment. Dual orexin antagonists may have a therapeutic potential in suppressing PTE potentially by enhancing GABAergic inhibition and impacting sleep homeostatic drive.

Evaluation of decompressive craniectomy in mice after severe traumatic brain injury

Decompressive craniectomy (DC) is of great significance for relieving acute intracranial hypertension and saving lives after traumatic brain injury (TBI). In this study, a severe TBI mouse model was created using controlled cortical impact (CCI), and a surgical model of DC was established. Furthermore, a series of neurological function assessments were performed to better understand the pathophysiological changes after DC. In this study, mice were randomly allocated into three groups, namely, CCI group, CCI+DC group, and Sham group. The mice in the CCI and CCI+DC groups received CCI after opening a bone window, and after brain injury, immediately returned the bone window to simulate skull condition after a TBI. The CCI+DC group underwent DC and contused tissue removal 6 h after CCI. The mice in the CCI group underwent the same anesthesia process; however, no further treatment of the bone window and trauma was performed. The mice in the Sham group underwent anesthesia and the process of opening the skin and bone window, but not in the CCI group. Changes in Modified Neurological Severity Score, rotarod performance, Morris water maze, intracranial pressure (ICP), cerebral blood flow (CBF), brain edema, blood–brain barrier (BBB), inflammatory factors, neuronal apoptosis, and glial cell expression were evaluated. Compared with the CCI group, the CCI+DC group had significantly lower ICP, superior neurological and motor function at 24 h after injury, and less severe BBB damage after injury. Most inflammatory cytokine expressions and the number of apoptotic cells in the brain tissue of mice in the CCI+DC group were lower than in the CCI group at 3 days after injury, with markedly reduced astrocyte and microglia expression. However, the degree of brain edema in the CCI+DC group was greater than in the CCI group, and neurological and motor functions, as well as spatial cognitive and learning ability, were significantly poorer at 14 days after injury.

Binary classification model of machine learning detected altered gut integrity in controlled-cortical impact model of traumatic brain injury

Aim of the study: To examine the effect of controlled-cortical impact (CCI), a preclinical model of traumatic brain injury (TBI), on intestinal integrity using a binary classification model of machine learning (ML). Materials and methods: Adult, male C57BL/6 mice were subjected to CCI surgery using a stereotaxic impactor (Impact One™). The rotarod and hot-plate tests were performed to assess the neurological deficits. Results: Mice underwent CCI displayed a remarkable neurological deficit as noticed by decreased latency to fall and lesser paw withdrawal latency in rotarod and hot plate test, respectively. Animals were sacrificed 3 days post-injury (dpi). The colon sections were stained with hematoxylin and eosin (H&E) to integrate with machinery tool-based algorithms. Several stained colon images were captured to build a dataset for ML model to predict the impact of CCI vs sham procedure. The best results were obtained with VGG16 features with SVM RBF kernel and VGG16 features with stacked fully connected layers on top. We achieved a test accuracy of 84% and predicted the disrupted gut permeability and epithelium wall of colon in CCI group as compared to sham-operated mice. Conclusion: We suggest that ML may become an important tool in the development of preclinical TBI model and discovery of newer therapeutics.

Determining Sex-Based Differences in Inflammatory Response in an Experimental Traumatic Brain Injury Model.

IntroductionTraumatic brain injury is a leading cause of injury-related death and morbidity. Multiple clinical and pre-clinical studies have reported various results regarding sex-based differences in TBI. Our accepted rodent model of traumatic brain injury was used to identify sex-based differences in the pathological features of TBI.MethodsMale and female Sprague-Dawley rats were subjected to either controlled-cortical impact (CCI) or sham injury; brain tissue was harvested at different time intervals depending on the specific study. Blood-brain barrier (BBB) analysis was performed using infrared imaging to measure fluorescence dye extravasation. Microglia and splenocytes were characterized with traditional flow cytometry; microglia markers such as CD45, P2Y12, CD32, and CD163 were analyzed with t-distributed stochastic neighbor embedding (t-SNE). Flow cytometry was used to study tissue cytokine levels, and supplemented with ELISAs of TNF-⍺, IL-17, and IL-1β of the ipsilateral hemisphere tissue.ResultsCCI groups of both sexes recorded a higher BBB permeability at 72 hours post-injury than their respective sham groups. There was significant difference in the integrated density value of BBB permeability between the male CCI group and the female CCI group (female CCI mean = 3.08 x 108 ± 2.83 x 107, male CCI mean = 2.20 x 108 ± 4.05 x 106, p = 0.0210), but otherwise no differences were observed. Traditional flow cytometry did not distinguish any sex-based difference in regards to splenocyte cell population after CCI. t-SNE did not reveal any significant difference between the male and female injury groups in the activation of microglia. Cytokine analysis after injury by flow cytometry and ELISA was limited in differences at the time point of 6 hours post-injury.ConclusionIn our rodent model of traumatic brain injury, sex-based differences in pathology and neuroinflammation at specified time points are limited, and only noted in one specific analysis of BBB permeability.

Quantitative Proteomics Reveals the Dynamic Pathophysiology Across Different Stages in a Rat Model of Severe Traumatic Brain Injury.

BackgroundSevere traumatic brain injury (TBI) has become a global health problem and causes a vast worldwide societal burden. However, distinct mechanisms between acute and subacute stages have not been systemically revealed. The present study aimed to identify differentially expressed proteins in severe TBI from the acute to subacute phase.MethodsSixty Sprague Dawley (SD) rats were randomly divided into sham surgery and model groups. The severe TBI models were induced by the controlled cortical impact (CCI) method. We evaluated the neurological deficits through the modified neurological severity score (NSS). Meanwhile, H&E staining and immunofluorescence were performed to assess the injured brain tissues. The protein expressions of the hippocampus on the wounded side of CCI groups and the same side of Sham groups were analyzed by the tandem mass tag-based (TMT) quantitative proteomics on the third and fourteenth days. Then, using the gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and protein–protein interaction (PPI), the shared and stage-specific differentially expressed proteins (DEPs) were screened, analyzed, and visualized. Eventually, target proteins were further verified by Western blotting (WB).ResultsIn the severe TBI, the neurological deficits always exist from the acute stage to the subacute stage, and brain parenchyma was dramatically impaired in either period. Of the significant DEPs identified, 312 were unique to the acute phase, 76 were specific to the subacute phase, and 63 were shared in both. Of the 375 DEPs between Sham-a and CCI-a, 240 and 135 proteins were up-regulated and down-regulated, respectively. Of 139 DEPs, 84 proteins were upregulated, and 55 were downregulated in the Sham-s and CCI-s. Bioinformatics analysis revealed that the differential pathophysiology across both stages. One of the most critical shared pathways is the complement and coagulation cascades. Notably, three pathways associated with gastric acid secretion, insulin secretion, and thyroid hormone synthesis were only enriched in the acute phase. Amyotrophic lateral sclerosis (ALS) was significantly enriched in the subacute stage. WB experiments confirmed the reliability of the TMT quantitative proteomics results.ConclusionOur findings highlight the same and different pathological processes in the acute and subacute phases of severe TBI at the proteomic level. The results of potential protein biomarkers might facilitate the design of novel strategies to treat TBI.

Sleep-wake characteristics in a mouse model of severe traumatic brain injury: Relation to posttraumatic epilepsy.

Study objectivesTraumatic brain injury (TBI) results in sequelae that include posttraumatic epilepsy (PTE) and sleep‐wake disturbances. Here, we sought to determine whether sleep characteristics could predict development of PTE in a model of severe TBI.MethodsFollowing controlled cortical impact (CCI) or sham injury (craniotomy only), CD‐1 mice were implanted with epidural electroencephalography (EEG) and nuchal electromyography (EMG) electrodes. Acute (1st week) and chronic (months 1, 2, or 3) 1‐week‐long video‐EEG recordings were performed after the injury to examine epileptiform activity. High‐amplitude interictal events were extracted from EEG using an automated method. After scoring sleep‐wake patterns, sleep spindles and EEG delta power were derived from nonrapid eye movement (NREM) sleep epochs. Brain CTs (computerized tomography) were performed in sham and CCI cohorts to quantify the brain lesions. We then employed a no craniotomy (NC) control to perform 1‐week‐long EEG recordings at week 1 and month 1 after surgery.ResultsPosttraumatic seizures were seen in the CCI group only, whereas interictal epileptiform activity was seen in CCI or sham. Sleep‐wake disruptions consisted of shorter wake or NREM bout lengths and shorter duration or lower power for spindles in CCI and sham. NREM EEG delta power increased in CCI and sham groups compared with NC though the CCI group with posttraumatic seizures had lower power at a chronic time point compared with those without. Follow‐up brain CTs showed a small lesion in the sham injury group suggesting a milder form of TBI that may account for their interictal activity and sleep changes.SignificanceIn our TBI model, tracking changes in NREM delta power distinguishes between CCI acutely and animals that will eventually develop PTE, but further work is necessary to identify sleep biomarkers of PTE. Employing NC controls together with sham controls should be considered in future TBI studies.

Germacrone alleviates neurological deficits following traumatic brain injury by modulating neuroinflammation and oxidative stress

BackgroundGermacrone (GM) is a terpenoid compound which is reported to have anti-inflammatory and anti-oxidative effects. However, its role in treating traumatic brain injury (TBI) remains largely unknown.MethodsMale C57BL/6 mice were divided into the following groups: control group, TBI group [controlled cortical impact (CCI) model], CCI + 5 mg/kg GM group, CCI + 10 mg/kg GM group and CCI + 20 mg/kg GM group. GM was administered via intraperitoneal injection. The neurological functions (including motor coordination, spatial learning and memory abilities) and brain edema were measured. Nissl staining was used to detect the neuronal apoptosis. Colorimetric assays and enzyme linked immunosorbent assay (ELISA) kits were used to determine the expression levels of oxidative stress markers including myeloperoxidase (MPO), malondialdehyde (MDA) and superoxide dismutase (SOD), as well as the expressions of inflammatory markers, including tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Additionally, protein levels of Nrf2 and p-p65 were detected by Western blot assay.ResultsGM significantly ameliorated motor dysfunction, spatial learning and memory deficits of the mice induced by TBI and it also reduced neuronal apoptosis and microglial activation in a dose-dependent manner. Besides, GM treatment reduced neuroinflammation and oxidative stress compared to those in the CCI group in a dose-dependent manner. Furthermore, GM up-regulated the expression of antioxidant protein Nrf2 and inhibited the expression of inflammatory response protein p-p65.ConclusionsGM is a promising drug to improve the functional recovery after TBI via repressing neuroinflammation and oxidative stress.

MiR-212-5p attenuates ferroptotic neuronal death after traumatic brain injury by targeting Ptgs2

Ferroptosis, a newly discovered form of iron-dependent regulated cell death, has been implicated in traumatic brain injury (TBI). MiR-212-5p has previously been reported to be downregulated in extracellular vesicles following TBI. To investigate whether miR-212-5p is involved in the ferroptotic neuronal death in TBI mice, we first examined the accumulation of malondialdehyde (MDA) and ferrous ion, and the expression of ferroptosis-related molecules at 6 h, 12 h, 24 h, 48 h and 72 h following controlled cortical impact (CCI) in mice. There was a significant upregulation in the expression of Gpx4 and Acsl4 at 6 h, Slc7a11 from 12 h to 72 h, and Nox2 and Sat1 from 6 h to 72 h post injury. Similarly, an upregulation in the expression of Gpx4 at 6 h, Nox2 from 6 h to 72 h, xCT from 12 h to 72 h, and Sat1 at 72 h after CCI was observed at the protein level. Interestingly, MDA and ferrous ion were increased whereas miR-212-5p was decreased in the CCI group compared to the sham group. Furthermore, we found that overexpression of miR-212-5p attenuated ferroptosis while downregulation of miR-212-5p promoted ferroptotic cell death partially by targeting prostaglandin-endoperoxide synthase-2 (Ptgs2) in HT-22 and Neuro-2a cell lines. In addition, administration of miR-212-5p in CCI mice significantly improved learning and spatial memory. Collectively, these findings indicate that miR-212-5p may protect against ferroptotic neuronal death in CCI mice partially by targeting Ptgs2.

The Recovery of GABAergic Function in the Hippocampus CA1 Region After mTBI.

Traumatic brain injury (TBI) is a major public health concern in the USA. There are approximately 2.5 million brain injuries annually, 90% of which may be classified as mild since these individuals do not display clear morphological abnormalities following injury on imaging. The majority of individuals develop neurocognitive deficits such as learning and memory impairment and recovery occurs over 3 to 6months after mild TBI (mTBI). The hippocampus is highly susceptible to injury from mTBI due to the anatomic localization and has been implicated in the neurocognitive impairments after mTBI. Here, we investigated whether the mTBI-induced morphological and pathophysiological alterations of GABAergic interneurons in the CA1 subfield of the hippocampus recovers after 30days in the controlled cortical impact (CCI) model of TBI. Design-based stereology shows a significant reduction in the number of GABAergic interneurons 7days after CCI. However, the number of GABAergic interneurons is not significantly reduced at 30days after CCI. The total number of neurons is not altered over the course of 30days. GABAergic inhibitory currents in the CA1 subfield also show that, although there is a significant reduction in the CCI group at 7days, the currents are not significantly different from sham controls at 30days. We suggest that the recovery of GABAergic function in the CA1 subfield of the hippocampus observed 30days after CCI is one of the mechanisms associated with the recovery of memory after mTBI.

Exploratory, cognitive, and depressive-like behaviors in adult and pediatric mice exposed to controlled cortical impact

ObjectiveSequelae of behavioral impairments associated with human traumatic brain injury (TBI) include neurobehavioral problems. We compared exploratory, cognitive, and depressive-like behaviors in pediatric and adult male mice exposed to controlled cortical impact (CCI).MethodsPediatric (21 to 25 days old) and adult (8 to 12 weeks old) male C57Bl/6 mice underwent CCI at a 2-mm depth of deflection. Hematoxylin and eosin staining was performed 3 to 7 days after recovery from CCI, and injury volume was analyzed using ImageJ. Neurobehavioral characterization after CCI was performed using the Barnes maze test (BMT), passive avoidance test, open-field test, light/dark test, tail suspension test, and rotarod test. Acutely and subacutely (3 and 7 days after CCI, respectively), CCI mice showed graded injury compared to sham mice for all analyzed deflection depths.ResultsTime-dependent differences in injury volume were noted between 3 and 7 days following 2-mm TBI in adult mice. In the BMT, 2-mm TBI adults showed spatial memory deficits compared to sham adults (P<0.05). However, no difference in spatial learning and memory was found between sham and 2-mm CCI groups among pediatric mice. The open-field test, light/dark test, and tail suspension test did not reveal differences in anxiety-like behaviors in both age groups.ConclusionOur findings revealed a graded injury response in both age groups. The BMT was an efficient cognitive test for assessing spatial/non-spatial learning following CCI in adult mice; however, spatial learning impairments in pediatric mice could not be assessed.

Intraparenchymal Application of Mature B Lymphocytes Improves Structural and Functional Outcome after Contusion Traumatic Brain Injury.

Cerebral contusion causes neurological dysfunction mediated in part by inflammatory responses to injury. B lymphocytes are dynamic regulators of the immune system that have not been systematically studied in traumatic brain injury (TBI). We showed previously that topically applied mature B cells have immunomodulatory properties and strongly promote tissue regeneration, including cutaneous nerve growth, in acute and chronic skin wounds. Using a mouse controlled cortical impact (CCI) model, we assessed a possible beneficial role of exogenously applied B cells on histopathological and functional outcome after TBI. Mice were injected intraparenchymally at the lesion site with 2 × 106 mature naïve syngeneic splenic B cells, then subjected to CCI. Control CCI mice received equal numbers of T cells or saline, and sham-injured mice (craniotomy only) were given B cells or saline. Sham-injured groups performed similarly in motor and learning tests. Injured mice administered B cells showed significantly improved post-injury rotarod, Y maze, and Morris water maze (MWM) performance compared with saline- or T-cell-treated CCI groups. Moreover, lesion volume in mice treated with B cells was significantly reduced by 40% at 35 days post-TBI compared with saline and T cell controls, and astrogliosis and microglial activation were decreased. In vivo tracking of exogenous B cells showed that they have a limited life span of approximately 14 days in situ and do not appear to proliferate. The data suggest proof of principle that local administration of B lymphocytes may represent a therapeutic option for treatment of cerebral contusion, especially when clinical management involves procedures that allow access to the injury site.

Focal traumatic brain injury induces neuroplastic molecular responses in lumbar spinal cord.

Background/Objectives:Motor impairment induced by traumatic brain injury (TBI) may be mediated through changes in spinal molecular systems regulating neuronal plasticity. We assessed whether a focal controlled cortical impact (CCI) TBI in the rat alters expression of the Tgfb1, c-Fos, Bdnf, and Gap43 neuroplasticity genes in lumbar spinal cord.Approach/Methods:Adult male Sprague-Dawley rats (n = 8) were subjected to a right-side CCI over the anterior sensorimotor hindlimb representation area or sham-injury (n = 8). Absolute expression levels of Tgfb1, c-Fos, Bdnf, and Gapd43 genes were measured by droplet digital PCR in ipsi-and contralesional, dorsal and ventral quadrants of the L4 and L5 spinal cord. The neuronal activity marker c-Fos was analysed by immunohistochemistry in the dorsal L4 and L5 segments. The contra- vs. ipsilesional expression pattern was examined as the asymmetry index, AI.Results:The Tgfb1 mRNA levels were significantly higher in the CCI vs. sham-injured rats, and in the contra- vs. ipsilesional dorsal domains in the CCI group. The number of c-Fos-positive cells was elevated in the L4 and L5 segments; and on the contralesional compared to the ipsilesional side in the CCI group. The c-Fos AI in the dorsal laminae was significantly increased by CCI.Conclusions:The results support the hypothesis that focal TBI induces plastic alterations in the lumbar spinal cord that may contribute to either motor recovery or maladaptive motor responses.