Animal model of repetitive mild traumatic brain injury for human traumatic axonal injury and chronic traumatic encephalopathy

Chronic traumatic encephalopathy（CTE）is a chronic neurodegenerative disease featured with tauopathy.CTE is tightly related with repetitive mild traumatic brain injury（m TBI）,which is interchangeably known as concussion（Mc Kee et al.,2009,2013）.This disease is differentiated by neuropathological features from other neurological diseases that involve tau protein aggregation and tangle formation abnormalities like Alzheimer＇s disease （AD）, frontotemporal dementia, and Parkinson- ism linked to chromosome 17 （FTDP-17）.

Stability of rat models of fluid percussion-induced traumatic brain injury: comparison of three different impact forces

Fluid percussion-induced traumatic brain injury models have been widely used in experimental research for years. In an experiment, the stability of impaction is inevitably affected by factors such as the appearance of liquid spikes. Management of impact pressure is a crucial factor that determines the stability of these models, and direction of impact control is another basic element. To improve experimental stability, we calculated a pressure curve by generating repeated impacts using a fluid percussion device at different pendulum angles. A stereotactic frame was used to control the direction of impact. We produced stable and reproducible models, including mild, moderate, and severe traumatic brain injury, using the MODEL01-B device at pendulum angles of 6°, 11° and 13°, with corresponding impact force values of 1.0 ± 0.11 atm（101.32 ± 11.16 k Pa）, 2.6 ± 0.16 atm（263.44 ± 16.21 k Pa）, and 3.6 ± 0.16 atm（364.77 ± 16.21 k Pa）, respectively. Behavioral tests, hematoxylin-eosin staining, and magnetic resonance imaging revealed that models for different degrees of injury were consistent with the clinical properties of mild, moderate, and severe craniocerebral injuries. Using this method, we established fluid percussion models for different degrees of injury and stabilized pathological features based on precise power and direction control.

The pig as a preclinical traumatic brain injury model:current models,functional outcome measures,and translational detection strategies

Traumatic brain injury(TBI) is a major contributor of long-term disability and a leading cause of death worldwide. A series of secondary injury cascades can contribute to cell death, tissue loss, and ultimately to the development of functional impairments. However, there are currently no effective therapeutic interventions that improve brain outcomes following TBI. As a result, a number of experimental TBI models have been developed to recapitulate TBI injury mechanisms and to test the efficacy of potential therapeutics. The pig model has recently come to the forefront as the pig brain is closer in size, structure, and composition to the human brain compared to traditional rodent models, making it an ideal large animal model to study TBI pathophysiology and functional outcomes. This review will focus on the shared characteristics between humans and pigs that make them ideal for modeling TBI and will review the three most common pig TBI models–the diffuse axonal injury, the controlled cortical impact, and the fluid percussion models. It will also review current advances in functional outcome assessment measures and other non-invasive, translational TBI detection and measurement tools like biomarker analysis and magnetic resonance imaging. The use of pigs as TBI models and the continued development and improvement of translational assessment modalities have made significant contributions to unraveling the complex cascade of TBI sequela and provide an important means to study potential clinically relevant therapeutic interventions.

Differences in pathological changes between two rat models of severe traumatic brain injury

The rat high-impact free weight drop model mimics the diffuse axonal injury caused by severe traumatic brain injury in humans,while severe controlled cortical impact can produce a severe traumatic brain injury model using precise strike parameters.In this study,we compare the pathological mechanisms and pathological changes between two rat severe brain injury models to identify the similarities and differences.The severe controlled cortical impact model was produced by an electronic controlled cortical impact device,while the severe free weight drop model was produced by dropping a 500 g free weight from a height of 1.8 m through a plastic tube.Body temperature and mortality were recorded,and neurological deficits were assessed with the modified neurological severity score.Brain edema and bloodbrain barrier damage were evaluated by assessing brain water content and Evans blue extravasation.In addition,a cytokine array kit was used to detect inflammatory cytokines.Neuronal apoptosis in the brain and brainstem was quantified by immunofluorescence staining.Both the severe controlled cortical impact and severe free weight drop models exhibited significant neurological impairments and body temperature fluctuations.More severe motor dysfunction was observed in the severe controlled cortical impact model,while more severe cognitive dysfunction was observed in the severe free weight drop model.Brain edema,inflammatory cytokine changes and cortical neuronal apoptosis were more substantial and blood-brain barrier damage was more focal in the severe controlled cortical impact group compared with the severe free weight drop group.The severe free weight drop model presented with more significant apoptosis in the brainstem and diffused blood-brain barrier damage,with higher mortality and lower repeatability compared with the severe controlled cortical impact group.Severe brainstem damage was not found in the severe controlled cortical impact model.These results indicate that the severe controlled cortical impact model is relatively more stable,more reproducible,and shows obvious cerebral pathological changes at an earlier stage.Therefore,the severe controlled cortical impact model is likely more suitable for studies on severe focal traumatic brain injury,while the severe free weight drop model may be more apt for studies on diffuse axonal injury.All experimental procedures were approved by the Ethics Committee of Animal Experiments of Tianjin Medical University,China(approval No.IRB2012-028-02)in Febru ary 2012.

MicroRNAs as diagnostic markers and therapeutic targets for traumatic brain injury

Traumatic brain injury （TBI） is characterized by primary damage to the brain from the external mechanical force and by subsequent secondary injury due to various molecular and pathophysiological responses that eventually lead to neuronal cell death. Secondary brain injury events may occur minutes, hours, or even days after the trauma, and provide valuable therapeutic targets to prevent further neuronal degeneration. At the present time, there is no effective treatment for TBI due, in part, to the widespread impact of numerous complex secondary biochemical and pathophysiological events occurring at different time points following the initial injury. MicroRNAs control a range of physiological and pathological functions such as develop- ment, differentiation, apoptosis and metabolism, and may serve as potential targets for progress assessment and intervention against TBI to mitigate secondary damage to the brain. This has implications regarding improving the diagnostic accuracy of brain impairment and long-term outcomes as well as potential novel treatments. Recent human studies have identified specific microRNAs in serum/plasma （miR-425-p, -21, -93, -191 and -499） and cerebro-spinal fluid （CSF） （miR-328, -362-3p, -451, -486a） as possible indicators of the diagnosis, severity, and prognosis of TBI. Experimental animal studies have examined specific microRNAs as biomarkers and therapeutic targets for moderate and mild TBI （e.g., miR-21, miR-23b）. MicroRNA profil- ing was altered by voluntary exercise. Differences in basal microRNA expression in the brain of adult and aged animals and alterations in response to TBI （e.g., miR-21） have also been reported. Further large-scale studies with TBI patients are needed to provide more information on the changes in microRNA profiles in different age groups （children, adults, and elderly）.

Microglia and astrocytes mediate synapse engulfment in a MER tyrosine kinase-dependent manner after traumatic brain injury

Recent studies have shown that microglia/macrophages and astrocytes can mediate synaptic phagocytosis through the MER proto-oncokinase in developmental or stroke models,but it is unclear whether the same mechanism is also active in traumatic brain injury.In this study,we established a mouse model of traumatic brain injury and found that both microglia/macrophages and astrocytes phagocytosed synapses and expression of the MER proto-oncokinase increased 14 days after injury.Specific knockout of MER in microglia/macrophages or astrocytes markedly reduced injury volume and greatly improved neurobehavioral function.In addition,in both microglia/macrophages-specific and astrocytes-specific MER knock-out mice,the number of microglia/macrophage and astrocyte phagocytosing synapses was markedly decreased,and the total number of dendritic spines was increased.Our study suggested that MER proto-oncokinase expression in microglia/macrophages and astrocytes may play an important role in synaptic phagocytosis,and inhibiting this process could be a new strategy for treating traumatic brain injury.

Altered expression of metabotropic glutamate receptor 1 alpha after acute diffuse brain injury Effect of the competitive antagonist 1-aminoindan-1, 5-dicarboxylic acid

The diffuse brain injury model was conducted in Sprague-Dawley rats, according to Marmarou＇s free-fall attack. The water content in brain tissue, expression of metabotropic glutamate receptor la mRNA and protein were significantly increased after injury, reached a peak at 24 hours, and then gradually decreased. After treatment with the competitive antagonist of metabotropic glutamate receptor la, （RS）-l-aminoindan-1,5-dicarboxylic acid, the water content of brain tissues decreased between 12-72 hours after injury, and neurological behaviors improved at 2 weeks. These experimental findings suggest that the 1-aminoindan-1, 5-dicarboxylic acid may result in marked neuroprotection against diffuse brain injury.

Establishment of a blunt impact-induced brain injury model in rabbits

Objective： To establish an animal model to replicate the blunt impact brain injury in forensic medicine. Methods： Twenty-four New Zealand white rabbits were randomly divided into control group （n=4）, minor injury group （n：10） and severe injury group （n=10）. Based on the BIM- II Horizontal Bio-impact Machine, self-designed iron bar was used to produce blunt brain injury. Two rabbits from each injury group were randomly selected to monitor the change ofintracranial pressure （ICP） during the impact- ing process by pressure microsensors. Six hours after injury, all the rabbits were dissected to observe the injury mor- phology and underwent routine pathological examination. Results： Varying degrees of nervous system positive signs were observed in all the injured rabbits. Within 6 hours, the mortality rate was 1/10 in the minor injury group and 6/10 in the severe injury group. Morphological changes con-sisted of different levels of scalp hematoma, skull fracture, epidural hematoma, subdural hematoma, subarachnoid hemo- rrhage and brain injury. At the moment of hitting, the ICP was greater in severe injury group than in mild injury group; and within the same group, the impact side showed positive pressure while the opposite side showed negative pressure. Conclusions： Under the rigidly-controlled experimental condition, this animal model has a good reproducibility and stable results. Meanwhile, it is able to simulate the morphology of iron strike-induced injury, thus can be used to study the mechanism of blunt head injury in forensic medicine.

Therapeutic effect of nimodipine on experimental brain injury

Objective: To study the therapeutic effect of nimodipine on experimental brain injury. Methods: Experimental and control rabbits were subjected to a closed head injury. In one group nimodipine was given intravenously and the effect evaluated by electron microscopy, brain water content, calcium levels, transcranial Doppler, and intracranial pressure monitoring. Results: In rabbits treated with nimodipine the level of neuronal cytosolic free calcium was markedly decreased. There were less cellular damage and less spasm of the middle cerebral artery seen on electron microscopy. No difference regarding intracranial pressure changes between the two groups was noted. Conclusions: Nimodipine has a protective action on brain injury by blocking a series of pathological reactions induced by neuronal calcium overload, and by reducing the spasm of brain vessels and improving cerebral blood flow.

Gliosis after traumatic brain injury in conditional ephrinB2-knockout mice

Background In response to the injury of the central nervous system （CNS）, the astrocytes upregulate the expression of glial fibrillary acidic protein （GFAP）, which largely contributes to the reactive gliosis after brain injury. The regulatory mechanism of this process is still not clear. In this study, we aimed to compare the ephrin-B2 deficient mice with the wild type ones with regard to qliosis after traumatic brain injury

MicroRNAs as disease progression biomarkers and therapeutic targets in experimental autoimmune encephalomyelitis model of multiple sclerosis

Multiple sclerosis is an autoimmune neurodegenerative disease of the central nervous system characterized by pronounced inflammatory infiltrates entering the brain,spinal cord and optic nerve leading to demyelination.Focal demyelination is associated with relapsing-remitting multiple sclerosis,while progressive forms of the disease show axonal degeneration and neuronal loss.The tests currently used in the clinical diagnosis and management of multiple sclerosis have limitations due to specificity and sensitivity.MicroRNAs(miRNAs)are dysregulated in many diseases and disorders including demyelinating and neuroinflammatory diseases.A review of recent studies with the experimental autoimmune encephalomyelitis animal model(mostly female mice 6–12 weeks of age)has confirmed miRNAs as biomarkers of experimental autoimmune encephalomyelitis disease and importantly at the pre-onset(asymptomatic)stage when assessed in blood plasma and urine exosomes,and spinal cord tissue.The expression of certain miRNAs was also dysregulated at the onset and peak of disease in blood plasma and urine exosomes,brain and spinal cord tissue,and at the post-peak(chronic)stage of experimental autoimmune encephalomyelitis disease in spinal cord tissue.Therapies using miRNA mimics or inhibitors were found to delay the induction and alleviate the severity of experimental autoimmune encephalomyelitis disease.Interestingly,experimental autoimmune encephalomyelitis disease severity was reduced by overexpression of miR-146a,miR-23b,miR-497,miR-26a,and miR-20b,or by suppression of miR-182,miR-181c,miR-223,miR-155,and miR-873.Further studies are warranted on determining more fully miRNA profiles in blood plasma and urine exosomes of experimental autoimmune encephalomyelitis animals since they could serve as biomarkers of asymptomatic multiple sclerosis and disease course.Additionally,studies should be performed with male mice of a similar age,and with aged male and female mice.

An animal model of cerebral palsy induced by prenatal exposure to lipopolysaccharide and hypoxia

BACKGROUND： Neonatal cerebral palsy is mainly caused by prenatal factors. At present, an animal model of prenatal infection and early postnatal hypoxia does not exist. OBJECTIVE： To observe morphology and motor performance following prenatal infection and hypoxic insult-induced brain damage of neonatal rats to verify the feasibility to establish a model of cerebral palsy. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Laboratories of Xinjiang Center for Disease Control and Prevention from September 2007 to June 2008. MATERIALS： The hypoxic incubator was purchased from Shanghai Pediatric Medical Institute, China. Lipopolysaccharide （LPS, Escherichia coil, 055： B5） was purchased from Sigma-Aldrich （St. Louis, MO, USA）. METHODS： A total of 27 Wistar rats, aged 7 days, were randomly assigned to sham-surgery group （n = 15） with no carotid artery incision or hypoxia treatment, hypoxia/ischemia （H/I） group （n = 12） undergoing ligature of the right common carotid artery followed by exposure to hypoxia at postnatal day 7 （P7）, and LPS/H group （n = 19）, in which pregnant rats were exposed in utero to LPS followed by prenatal hypoxia at embryonic day 16. MAIN OUTCOME MEASURES： Behavior, compound muscle action potential, and pathological changes were observed in 28-day-old rats. RESULTS： The footprint repeat space showed that left limb footprint repeatability in the H/I and LPS/H groups was lower than in the sham-surgery group （P 〈 0.05）. The space between the footprints was larger and unstable. Hind limb quadricep compound muscle action potential in the H/I and LPS/H groups showed lower wave amplitude compared with the sham-surgery group （P〈 0.05） Hematoxylin and eosin staining showed irregular cells around the ventricle, as well as periventricular leukomalacia. CONCLUSION： An animal model of cerebral palsy was established, which simulated the human condition most likely associated with occurrence of this disease. This model could be used for experimental studies related to cerebral palsy.

Relationship between AQP4 expression and structural damage to the blood-brain barrier at early stages of traumatic brain injury in rats

Background Although some studies have reported that aquaporin-4 （AQP4） plays an important role in the brain edema after traumatic brain injury （TBI）, little is known about the AQP4 expression in the early stage of TBI, or about the correlation between the structural damage to the blood-brain barrier （BBB） and angioedema. The aim of this project was to investigate the relationship between AQP4 expression and damage to the BBB at early stages of TBI. Methods One hundred and twenty healthy adult Wistar rats were randomly divided into two groups： sham operation group （SO） and TBI group. The TBI group was divided into five sub-groups according to the different time intervals： 1, 3, 6, 12, and 24 hours. The brains of the animals were taken out at different time points after TBI to measure brain water content. The cerebral edema and BBB changes in structure were examined with an optical microscopy （OM） and transmission electron microscopy （TEM）, and the IgG content and AQP4 protein expression in traumatic brain tissue were determined by means of immunohistochemistry and Western blotting. The data were analyzed with SPSS 13.0 statistical software. Results In the SO group, tissue was negative for IgG, and there were no abnormalities in brain water content or AQP4 expression. In the TBI group, brain water content significantly increased at 6 hours and peaked at 24 hours following injury. IgG expression significantly increased from 1 to 6 hours following injury, and remained at a high level at 24 hours. Pathological observation revealed BBB damage at 1 hour following injury. Angioedema appeared at 1 hour, was gradually aggravated, and became obvious at 6 hours. Intracellular edema occurred at 3 hours, with the presence of large glial cell bodies and mitochondrial swelling. These phenomena were aggravated with time and became obvious at 12 hours. In addition, microglial proliferation was visible at 24 hours. AQP4 protein expression were reduced at 1 hour, lowest at 6 hours, and began to increase at 12 hours, showing a V-shaped curve. Conclusions The angioedema characterized by BBB damage was the primary type of early traumatic brain edema. It was followed by mixed cerebral edema that consisted of angioedema and cellular edema and was aggravated with time. AQP4 expression was down-regulated during the angioedema attack, but AQP4 expression was upregulated during intracellular edema.

An Experimental Study of Craniocerebral Injury Caused by 7. 62 mm Bullets in Dogs

The experimental models of craniocercbral wounds caused by 7. 62 mm bullets, i. e. thepenetrating craniocerebral injury, the tangential brain injury and the tangential skull injury, were es-tablished in dogs. The craniocerebral ballistics, craniocerebral pathology, serum and cerebrospinal flu-id total lactate dehydrogenase, blood-brain barrier permcabalities, and the pathophysiology ofcardiovascular and respiratory systems were studied. These results suggest that: 1. These injuries ofhigh-velocity missile can all cause general brain damage and intracranio-hematomas ; 2. The severityof the wound depends on the site of the injury, the kinetic energy of the missile force and the effectof the temporary cavity ; 3. The brain injury can seriously damage the blood brain barrier, leadingto brain edema ; 4. The dysfunction of respiratory and cardiovascular system is the fatal complicationendangering the life of the subjects ; 5. Estimating serum and cerebrospinal fluid total lactatedehydrogenase is a simple and valuable way to judge the severity and prognosis of this injury.

An Experimental Study on the Treatment with High Dose Dexamethasone of Craniocerebral Injury Caused by 7.62mm Bullets in Dogs

The effect of high dose dexamethasone(5 mg/kg wt,intravenous injection)to preventand treat secondary pathological damage due to craniocerebral injury was studied in an animal modelof craniocerebral injury caused by high-velocity missiles in dogs.We observed the physiologicalchanges,analyzed the level of serum and cerebrospinal fluid lactate dehydrogenase,cstimated thepermeability of blood brain barrier(BBB)and studied brain pathology by light and electronmicroscopy.The rusults suggest that high dose dexarnethasone can help to restore the structure andfunction of BBB,protect the brain cells,lessen the secondary pathological damage in the respiratoryand circulatory systams,and reduce the production of lipoperoxides(LPO).

Establishing a rat model of spastic cerebral palsy by targeted ethanol injection

Spastic cerebral palsy is generally considered to result from cerebral cortical or pyramidal tract damage. Here, we precisely targeted the left pyramidal tract of 2-month-old Sprague-Dawley rats placed on a stereotaxic instrument under intraperitoneal anesthesia. Based on the rat brain stereotaxic map, a 1-mm hole was made 10 mm posterior to bregma and 0.8 mm left of sagittal suture. A microsyringe was inserted perpendicularly to the surface of the brain to a depth of 9.7 mm, and 15 wL of ethanol was slowly injected to establish a rat model of spastic cerebral palsy. After modeling, the rats appeared to have necrotic voids in the pyramidal tract and exhibited typical signs and symptoms of flexion spasms that lasted for a long period of time. These findings indicate that this is an effective and easy method of establishing a rat model of spastic cerebral palsy with good reproducibility. Ethanol as a chemical ablation agent specifically and thoroughly damages the py- ramidal tract, and therefore, the animals display flexion spasms, which are a typical symptom of the disease.

Nanometer ultrastructural brain damage following low intensity primary blast wave exposure

Blast-induced mild traumatic brain injury（m TBI） is of particular concern among military personnel due to exposure to blast energy during military training and combat.The impact of primary low-intensity blast mediated pathophysiology upon later neurobehavioral disorders has been controversial.Developing a military preclinical blast model to simulate the pathophysiology of human blast injury is an important first step.This article provides an overview of primary blast effects and perspectives of our recent studies demonstrating ultrastructural changes in the brain and behavioral disorders resulting from open-field blast exposures up to 46.6 k Pa using a murine model.The model is scalable and permits exposure to varying magnitudes of primary blast injuries by placing animals at different distances from the blast center or by changing the amount of C4 charge.We here review the implications and future applications and directions of using this animal model to uncover the underlying mechanisms related to primary blast injury.Overall,these studies offer the prospect of enhanced understanding of the pathogenesis of primary low-intensity blast-induced TBI and insights for prevention,diagnosis and treatment of blast induced TBI,particularly m TBI/concussion related to current combat exposures.

How does the motor relearning program improve neurological function of brain ischemia monkeys?

The motor relearning program can significantly improve various functional disturbance induced by ischemic cerebrovascular diseases. However, its mechanism of action remains poorly understood. In injured brain tissues, glial fibrillary acidic protein and neurofilament protein changes can reflect the condition of injured neurons and astrocytes, while vascular endothelial growth factor and basic fibroblast growth factor changes can indicate angiogenesis. In the present study, we induced ischemic brain injury in the rhesus macaque by electrocoagulation of the M1 segment of the right middle cerebral artery. The motor relearning program was conducted for 60 days from the third day after model establishment. Immunohistochemistry and single-photon emission CT showed that the numbers of glial fibrillary acidic protein-, neurofilament protein-, vascular endothelial growth factor- and basic fibroblast growth factor-positive cells were significantly increased in the infarcted side compared with the contralateral hemisphere following the motor relearning program. Moreover, cerebral blood flow in the infarcted side was significantly improved. The clinical rating scale for stroke was used to assess neurological function changes in the rhesus macaque following the motor relearning program. Results showed that motor function was improved, and problems with consciousness, self-care ability and balance function were significantly ameliorated. These findings indicate that the motor relearning program significantly promoted neuronal regeneration, repair and angiogenesis in the surroundings of the infarcted hemisphere, and improve neurological function in the rhesus macaque following brain ischemia.

Hydrogen sulfide intervention in focal cerebral ischemia/reperfusion injury in rats

The present study aimed to explore the mechanism underlying the protective effects of hydrogen sulfide against neuronal damage caused by cerebral ischemia/reperfusion. We established the middle cerebral artery occlusion model in rats via the suture method. Ten minutes after middle cerebral artery occlusion, the animals were intraperitoneally injected with hydrogen sulfide donor compound sodium hydrosulfide. Immunofluorescence revealed that the immunoreactivity of P2X7 in the cerebral cortex and hippocampal CA1 region in rats with cerebral ischemia/reperfusion injury decreased with hydrogen sulfide treatment. Furthermore, treatment of these rats with hydrogen sulfide significantly lowered mortality, the Longa neurological deficit scores, and infarct volume. These results indicate that hydrogen sulfide may be protective in rats with local cerebral ischemia/reperfusion injury by down-regulating the expression of P2X7 receptors.

A rat pup model of cerebral palsy induced by prenatal inflammation and hypoxia

Animal models of cerebral palsy established by simple infection or the hypoxia/ischemia method cannot effectively simulate the brain injury of a premature infant. Healthy 17-day-pregnant Wistar rats were intraperitoneally injected with lipopolysaccharide then subjected to hypoxia. The pups were used for this study at 4 weeks of age. Simultaneously, a hypoxia/ischemia group and a control group were used for comparison. The results of the footprint test, the balance beam test, the water maze test, neuroelectrophysiological examination and neuropathological examination demonstrated that, at 4 weeks after birth, footprint repeat space became larger between the forelimbs and hindlimbs of the rats, the latency period on the balance beam and in the Morris water maze was longer, place navigation and ability were poorer, and the stimulus intensity that induced the maximal wave amplitude of the compound muscle action potential was greater in the lipopolysaccharide/hypoxia and hypoxia/ischemia groups than in the control group. We observed irregular cells around the periventricular area, periventricular leukomalacia and breakage of the nuclear membrane in the lipopolysacchadde/hypexia and hypoxia/ischemia groups. These results indicate that we successfully established a Wistar rat pup model of cerebral palsy by intraperitoneal injection of lipopolysaccharide and hypoxia.