Brain trauma and autophagy: What flies and mice can teach us about conserved responses

Drosophila models have been successfully used to identify many genetic components that affect neurodegenerative disorders. Recently, there has been a growing interest in identifying innate and environmental factors that influence the individual outcomes following traumatic brain injury (TBI). This includes both severe TBI and more subtle, mild TBI (mTBI), which is common in people playing contact sports. Autophagy, as a clearance pathway, exerts protective effects in multiple neurological disease models. In a recent publication, we highlighted the development of a novel repetitive mTBI system using Drosophila, which recapitulates several phenotypes associated with trauma in mammalian models. In particular, flies subjected to mTBI exhibit an acute impairment of the macroautophagy/autophagy pathway that is restored 1 wk following traumatic injury exposure. These phenotypes closely resemble temporary autophagy defects observed in a mouse TBI model. Through these studies, we also identified methods to directly assess autophagic responses in the fly nervous system and laid the groundwork for future studies designed to identify genetic, epigenetic and environmental factors that have an impact on TBI outcomes.     

A literature review of the feasibility of glial fibrillary acidic protein as a biomarker for stroke and traumatic brain injury

Traumatic brain injuries (TBIs) are potentially lethal medical conditions, with symptoms that can overlap with symptoms of injuries outside the brain. In many cases, current diagnostic methods do not fully distinguish acute brain injury from other organ damage. In the management of stroke patients, the choice of treatment depends on whether the stroke is ischemic or hemorrhagic; however, no quick lab diagnostic tests are available to distinguish between the two types of strokes. As a result, patient triage, disposition, and patient management decisions may be delayed for patients with suspected TBI and stroke. Glial fibrillary acidic protein (GFAP), a brain-specific biomarker that is released into the blood following TBI and stroke, is being explored for potential diagnostic and prognostic value in these indications. We therefore conducted a review of MEDLINE-indexed publications from 2004 to 2011 to evaluate the current status of GFAP as a prognostic and diagnostic tool for TBI and stroke within the context of current published guidelines. Our review suggests that GFAP could provide clinically valuable information for the prognosis of TBI and stroke, but it is still at an early stage of development as a biomarker. Several TBI studies have shown elevated GFAP levels following a TBI event to be associated with greater severity of injury, poorer outcomes, and increased mortality. Clinical studies also indicate that GFAP has potential clinical utility in the differential diagnosis of various types of stroke. However, more clinical research will be required to determine the ability of GFAP levels to diagnose TBI in heterogeneous patient populations, as well as the ability of GFAP to differentiate between ischemic stroke (IS), intracerebral hemorrhage (ICH), subarachnoid hemorrhage (SAH), and non-stroke conditions in populations of patients with suspected rather than confirmed stroke. Additional clinical studies will also be required to define the temporal patterns of GFAP release in IS, ICH, SAH, and TBI, and their potential use in the differential diagnosis of these conditions. Finally, such research could demonstrate the ability of GFAP test results to provide unique clinical information that informs management decisions for TBI and stroke patients.     

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.     

Role of the Prostaglandin E2 EP1 Receptor in Traumatic Brain Injury

Brain injuries promote upregulation of so-called proinflammatory prostaglandins, notably prostaglandin E₂ (PGE₂), leading to overactivation of a class of its cognate G-protein-coupled receptors, including EP1, which is considered a promising target for treatment of ischemic stroke. However, the role of the EP1 receptor is complex and depends on the type of brain injury. This study is focused on the investigation of the role of the EP1 receptor in a controlled cortical impact (CCI) model, a preclinical model of traumatic brain injury (TBI). The therapeutic effects of post-treatments with a widely studied EP1 receptor antagonist, SC-51089, were examined in wildtype and EP1 receptor knockout C57BL/6 mice. Neurological deficit scores (NDS) were assessed 24 and 48 h following CCI or sham surgery, and brain immunohistochemical pathology was assessed 48 h after surgery. In wildtype mice, CCI resulted in an obvious cortical lesion and localized hippocampal edema with an associated significant increase in NDS compared to sham-operated animals. Post-treatments with the selective EP1 receptor antagonist SC-51089 or genetic knockout of EP1 receptor had no significant effects on cortical lesions and hippocampal swelling or on the NDS 24 and 48 h after CCI. Immunohistochemistry studies revealed CCI-induced gliosis and microglial activation in selected ipsilateral brain regions that were not affected by SC-51089 or in the EP1 receptor-deleted mice. This study provides further clarification on the respective contribution of the EP1 receptor in TBI and suggests that, under this experimental paradigm, the EP1 receptor would have limited effects in modulating acute neurological and anatomical pathologies following contusive brain trauma. Findings from this protocol, in combination with previous studies demonstrating differential roles of EP1 receptor in ischemic, neurotoxic, and hemorrhagic conditions, provide scientific background and further clarification of potential therapeutic application of prospective prostaglandin G-protein-coupled receptor drugs in the clinic for treatment of TBI and other acute brain injuries.     

Protection against radiation-induced bone marrow and intestinal injuries by Cordyceps sinensis, a Chinese herbal medicine

Bone marrow and intestinal damage limits the efficacy of radiotherapy for cancer and can result in death if the whole body is exposed to too high a dose, as might be the case in a nuclear accident or terrorist incident. Identification of an effective nontoxic biological radioprotector is therefore a matter of some urgency. In this study, we show that an orally administered hot-water extract from a Chinese herbal medicine, Cordyceps sinensis (CS), protects mice from bone marrow and intestinal injuries after total-body irradiation (TBI). CS increased the median time to death from 13 to 20 days after 8 Gy TBI and from 9 to 18 days after 10 Gy TBI. Although CS-treated mice receiving 10 Gy TBI survived intestinal injury, most died from bone marrow failure, as shown by severe marrow hypoplasia in mice dying between 18 and 24 days. At lower TBI doses of 5.5 and 6.5 Gy, CS protected against bone marrow death, an effect that was confirmed by the finding that white blood cell counts recovered more rapidly. In vitro, CS reduced the levels of free radical species (ROS) within cells, and this is one likely mechanism for the radioprotective effects of CS, although probably not the only one.     

Island phytophagy: explaining the remarkable diversity of plant-feeding insects

Plant-feeding insects have undergone unparalleled diversification among different plant taxa, yet explanations for variation in their diversity lack a quantitative, predictive framework. Island biogeographic theory has been applied to spatially discrete habitats but not to habitats, such as host plants, separated by genetic distance. We show that relationships between the diversity of gall-inducing flies and their host plants meet several fundamental predictions from island biogeographic theory. First, plant-taxon genetic distinctiveness, an integrator for long-term evolutionary history of plant lineages, is a significant predictor of variance in the diversity of gall-inducing flies among host-plant taxa. Second, range size and structural complexity also explain significant proportions of the variance in diversity of gall-inducing flies among different host-plant taxa. Third, as with other island systems, plant-lineage age does not predict species diversity. Island biogeographic theory, applied to habitats defined by genetic distance, provides a novel, comprehensive framework for analysing and explaining the diversity of plant-feeding insects and other host-specific taxa.     

Evolutionary ecology of intraspecific brain size variation: a review

The brain is a trait of central importance for organismal performance and fitness. To date, evolutionary studies of brain size variation have mainly utilized comparative methods applied at the level of species or higher taxa. However, these studies suffer from the difficulty of separating causality from correlation. In the other extreme, studies of brain plasticity have focused mainly on within‐population patterns. Between these extremes lie interpopulational studies, focusing on brain size variation among populations of the same species that occupy different habitats or selective regimes. These studies form a rapidly growing field of investigations which can help us to understand brain evolution by providing a test bed for ideas born out of interspecific studies, as well as aid in uncovering the relative importance of genetic and environmental factors shaping variation in brain size and architecture. Aside from providing the first in depth review of published intraspecific studies of brain size variation, we discuss the prospects embedded with interpopulational studies of brain size variation. In particular, the following topics are identified as deserving further attention: (i) studies focusing on disentangling the contributions of genes, environment, and their interactions on brain variation within and among populations, (ii) studies applying quantitative genetic tools to evaluate the relative importance of genetic and environmental factors on brain features at different ontogenetic stages, (iii) apart from utilizing simple gross estimates of brain size, future studies could benefit from use of neuroanatomical, neurohistological, and/or molecular methods in characterizing variation in brain size and architecture.     

重度创伤性脑损伤后肠黏膜屏障应激性变化的模型

目的建立一种观察重度创伤性脑损伤(TBI)后肠黏膜屏障(IMB)应激性变化的模型。方法选用雄性Wistar大鼠64只,随机分为两组。TBI组(32只):采用改良的Feeney自由落体撞击法,建立TBI模型;假手术对照组(32只):只开骨窗,不行落体致伤。两组大鼠分别按术后6、12、24和48h时相点分为4个亚组(每组均为8只),观察脑组织、肠黏膜组织病理以及扫描和透射电镜下肠黏膜超微结构的变化。结果光镜下TBI组肠黏膜上皮细胞受损,电镜下还可见细胞间紧密连接较对照组明显增宽。结论用改良的Feeney自由落体撞击法,建立的重度TBI大鼠模型肠黏膜上皮细胞受损,细胞间紧密连接增宽,提示其IMB的功能的确发生了应激性损害,说明这种用来观察重度TBI后IMB应激性变化的模型是成功的。     

Neuroregenerative strategies in the brain: emerging significance of bone morphogenetic protein 7 (BMP7)

Every year thousands of people suffer from brain injuries and stroke, and develop motor, sensory, and cognitive problems as a result of neuronal loss in the brain. Unfortunately, the damaged brain has a limited ability to enact repair and current modes of treatment are not sufficient to offset the damage. An extensive list of growth factors, neurotrophic factors, cytokines, and drugs has been explored as potential therapies. However, only a limited number of them may actually have the potential to effectively offset the brain injury or stroke-related problems. One of the treatments considered for future brain repair is bone morphogenetic protein 7 (BMP7), a factor currently used in patients to treat non-neurological diseases. The clinical application of BMP7 is based on its neuroprotective role in stroke animal models. This paper reviews the current approaches considered for brain repair and discusses the novel convergent strategies by which BMP7 potentially can induce neuroregeneration.     

The mitochondrial permeability transition pore provides a key to the diagnosis and treatment of traumatic brain injury

The pathological consequences of traumatic head injury result largely from the opening of the mitochondrial permeability transition pore (mPTP). The mPTP opens due to a decrease in brain phosphorylation energy resulting in a further decrease in brain ATP production and a measurable increase in brain heat generation and temperature. The increase in brain temperature can be measured transcranially by near infrared spectroscopy which can be used to diagnose traumatic brain injury (TBI) and to monitor treatment. Effective therapy of TBI can be achieved by closure of the mPTP by administration of cyclosporine A or by oral administration of ketone body esters. While ketosis has previously been known to prevent damage from TBI, the availability of oral ketone esters presents the first practical modality of achieving therapeutic levels of ketone bodies.     

The protective effect of alpha lipoic acid against traumatic brain injury in rats

Traumatic brain injury (TBI) was induced by a weight-drop device using 300 g–1 m weight-height impact. The study groups were: control, alpha-lipoic acid (LA) (100 mg/kg, po), TBI, and TBI + LA (100 mg/kg, po). Forty-eight hours after the injury, neurological scores were measured and brain samples were taken for histological examination or determination of thiobarbituric acid reactive substances (TBARS) and glutathione (GSH) levels, myeloperoxidase (MPO) and Na⁺-K⁺ ATPase activities, whereas cytokines (TNF-α, IL-1β) were determined in blood. Brain oedema was evaluated by wet–dry weight method and blood–brain barrier (BBB) permeability was evaluated by Evans Blue (EB) extravasation. As a result, neurological scores mildly increased in trauma groups. Moreover, TBI caused a significant decrease in brain GSH and Na⁺-K⁺ ATPase activity, which was accompanied with significant increases in TBARS level, MPO activity and plasma proinflammatory cytokines. LA treatment reversed all these biochemical indices as well as histopathological alterations. TBI also caused a significant increase in brain water content and EB extravasation which were partially reversed by LA treatment. These findings suggest that LA exerts neuroprotection by preserving BBB permeability and by reducing brain oedema probably by its anti-inflammatory and antioxidant properties in the TBI model.     

Reactive Oxygen Species-Activated Nanoprodrug of Ibuprofen for Targeting Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) is an enormous public health problem, with 1.7 million new cases of TBI recorded annually by the Centers for Disease Control. However, TBI has proven to be an extremely challenging condition to treat. Here, we apply a nanoprodrug strategy in a mouse model of TBI. The novel nanoprodrug contains a derivative of the nonsteroidal anti-inflammatory drug (NSAID) ibuprofen in an emulsion with the antioxidant α-tocopherol. The ibuprofen derivative, Ibu₂TEG, contains a tetra ethylene glycol (TEG) spacer consisting of biodegradable ester bonds. The biodegradable ester bonds ensure that the prodrug molecules break down hydrolytically or enzymatically. The drug is labeled with the fluorescent reporter Cy5.5 using nonbiodegradable bonds to 1-octadecanethiol, allowing us to reliably track its accumulation in the brain after TBI. We delivered a moderate injury using a highly reproducible mouse model of closed-skull controlled cortical impact to the parietal region of the cortex, followed by an injection of the nanoprodrug at a dose of 0.2 mg per mouse. The blood brain barrier is known to exhibit increased permeability at the site of injury. We tested for accumulation of the fluorescent drug particles at the site of injury using confocal and bioluminescence imaging of whole brains and brain slices 36 hours after administration. We demonstrated that the drug does accumulate preferentially in the region of injured tissue, likely due to an enhanced permeability and retention (EPR) phenomenon. The use of a nanoprodrug approach to deliver therapeutics in TBI represents a promising potential therapeutic modality.     

Valproic Acid: A New Candidate of Therapeutic Application for the Acute Central Nervous System Injuries

Acute central nervous system (CNS) injuries, including stroke, traumatic brain injury (TBI), and spinal cord injury (SCI), are common causes of human disabilities and deaths, but the pathophysiology of these diseases is not fully elucidated and, thus, effective pharmacotherapies are still lacking. Valproic acid (VPA), an inhibitor of histone deacetylation, is mainly used to treat epilepsy and bipolar disorder with few complications. Recently, the neuroprotective effects of VPA have been demonstrated in several models of acute CNS injuries, such as stroke, TBI, and SCI. VPA protects the brain from injury progression via anti-inflammatory, anti-apoptotic, and neurotrophic effects. In this review, we focus on the emerging neuroprotective properties of VPA and explore the underlying mechanisms. In particular, we discuss several potential related factors in VPA research and present the opportunity to administer VPA as a novel neuropective agent.     

Ghrelin attenuates brain injury after traumatic brain injury and uncontrolled hemorrhagic shock in rats

Traumatic brain injury (TBI) and hemorrhagic shock often occur concomitantly due to multiple injuries. Gastrointestinal dysfunction occurs frequently in patients with TBI. However, whether alterations in the gastrointestinal system are involved in modulating neuronal damage and recovery after TBI is largely neglected. Ghrelin is a "gut-brain" hormone with multiple functions including antiinflammation and antiapoptosis. The purpose of this study was to determine whether ghrelin attenuates brain injury in a rat model of TBI and uncontrolled hemorrhage (UH). To study this, brain injury was induced by dropping a 450-g weight from 1.5 m onto a steel helmet attached to the skull of male adult rats. Immediately after TBI, a midline laparotomy was performed and both lumbar veins were isolated and severed at the junction with the vena cava. At 45 min after TBI/UH, ghrelin (4, 8 or 16 nmol/rat) or 1 mL normal saline (vehicle) was intravenously administered. Brain levels of TNF-α and IL-6, and cleaved PARP-1 levels in the cortex were measured at 4 h after TBI/UH. Beam balance test, forelimb placing test and hindlimb placing test were used to assess sensorimotor and reflex function. In additional groups of animals, ghrelin (16 nmol/rat) or vehicle was subcutaneously (s.c.) administered daily for 10 d after TBI/UH. The animals were monitored for 28 d to record body weight changes, neurological severity scale and survival. Our results showed that ghrelin downregulated brain levels of TNF-α and IL-6, reduced cortical levels of cleaved PARP-1, improved sensorimotor and reflex functions, and decreased mortality after TBI/UH. Thus, ghrelin has a great potential to be further developed as an effective resuscitation approach for the trauma victims with brain injury and severe blood loss.     

The Effects of Lactobacillus acidophilus on the Intestinal Smooth Muscle Contraction through PKC/MLCK/MLC Signaling Pathway in TBI Mouse Model

Clinical studies have shown that probiotics influence gastrointestinal motility. However, the molecular mechanisms by which probiotic Lactobacillus modulates intestinal motility in traumatic brain injury (TBI) mouse model have not been explored. In the present study, we provided evidence showing that treatment of TBI mice with Lactobacillus acidophilus significantly improved the terminal ileum villus morphology, restored the impaired interstitial cells of Cajal (ICC) and the disrupted ICC networks after TBI, and prevented TBI-mediated inhibition of contractile activity in intestinal smooth muscle. Mechanistically, the decreased concentration of MLCK, phospho-MLC₂₀ and phospho-MYPT1 and increased concentration of MLCP and PKC were observed after TBI, and these events mediated by TBI were efficiently prevented by Lactobacillus acidophilus application. These findings may provide a novel mechanistic basis for the application of Lactobacillus acidophilus in the treatment of TBI.     

Temporal and spatial profile of caspase 8 expression and proteolysis after experimental traumatic brain injury

Recent studies have demonstrated that the downstream caspases, such as caspase 3, act as executors of the apoptotic cascade after traumatic brain injury (TBI) in vivo. However, little is known about the involvement of caspases in the initiation phase of apoptosis, and the interaction between these initiator caspases (e.g. caspase 8) and executor caspases after experimental brain injuries in vitro and in vivo. This study investigated the temporal expression and cell subtype distribution of procaspase 8 and cleaved caspase 8 p20 from 1 h to 14 days after cortical impact-induced TBI in rats. Caspase 8 messenger RNA levels, estimated by semiquantitaive RT-PCR, were elevated from 1 h to 72 h in the traumatized cortex. Western blotting revealed increased immunoreactivity for procaspase 8 and the proteolytically active subunit of caspase 8, p20, in the ipsilateral cortex from 6 to 72 h after injury, with a peak at 24 h after TBI. Similar to our previous studies, immunoreactivity for the p18 fragment of activated caspase 3 also increased in the current study from 6 to 72 h after TBI, but peaked at a later timepoint (48 h) as compared with proteolyzed caspase 8 p20. Immunohistologic examinations revealed increased expression of caspase 8 in neurons, astrocytes and oligodendrocytes. Assessment of DNA damage using TUNEL identified caspase 8- and caspase 3-immunopositive cells with apoptotic-like morphology in the cortex ipsilateral to the injury site, and immunohistochemical investigations of caspase 8 and activated caspase 3 revealed expression of both proteases in cortical layers 2-5 after TBI. Quantitative analysis revealed that the number of caspase 8 positive cells exceeds the number of caspase 3 expressing cells up to 24 h after impact injury. In contrast, no evidence of caspase 8 and caspase 3 activation was seen in the ipsilateral hippocampus, contralateral cortex and hippocampus up to 14 days after the impact. Our results provide the first evidence of caspase 8 activation after experimental TBI and suggest that this may occur in neurons, astrocytes and oligodendrocytes. Our findings also suggest a contributory role of caspase 8 activation to caspase 3 mediated apoptotic cell death after experimental TBI in vivo.     

Involvement of Connexin40 in the Protective Effects of Ginsenoside Rb1 Against Traumatic Brain Injury

Ginsenosides are the major active components of ginseng, which have been proven to be effective in therapies for neurodegenerative diseases. Ginsenoside Rb1 (GS-Rb1) is the most abundant among all the identified ginsenosides and has been shown to exert neuroprotective effects, although the underlying molecular mechanisms remain unclear. Connexins are a family of transmembrane proteins that form gap junctions, which are important for diffusion of cytosolic factors such as ions and second messenger signaling molecules. Previous studies have shown that a subset of connexin proteins is involved in neuroprotection. We investigated the protective effects of GS-Rb1 against traumatic brain injury (TBI) and the potential mechanism using TBI mouse model. We discovered that TBI-induced brain injury and up-regulation of connexin40 (Cx40) protein expression as early as 6 h post-TBI, which was reversed by administration of GS-Rb1. In addition, we found that the protective effects of GS-Rb1 are dose and time dependent and are partially mediated through phosphorylation of ERK1/2 signaling pathway, as evidenced by the abolishment of GS-Rb1-mediated elevation of p-ERK1/2 expression and inhibition of Cx40 expressions when ERK inhibitor U0126 was used. Our study provides evidence that Cx40 is implicated in TBI-induced brain injuries, and GS-Rb1 exerts neuroprotective activity against TBI involving down-regulation of Cx40 expression.     

Addressing the needs of traumatic brain injury with clinical proteomics

Background

Neurotrauma or injuries to the central nervous system (CNS) are a serious public health problem worldwide. Approximately 75% of all traumatic brain injuries (TBIs) are concussions or other mild TBI (mTBI) forms. Evaluation of concussion injury today is limited to an assessment of behavioral symptoms, often with delay and subject to motivation. Hence, there is an urgent need for an accurate chemical measure in biofluids to serve as a diagnostic tool for invisible brain wounds, to monitor severe patient trajectories, and to predict survival chances. Although a number of neurotrauma marker candidates have been reported, the broad spectrum of TBI limits the significance of small cohort studies. Specificity and sensitivity issues compound the development of a conclusive diagnostic assay, especially for concussion patients. Thus, the neurotrauma field currently has no diagnostic biofluid test in clinical use.

Content

We discuss the challenges of discovering new and validating identified neurotrauma marker candidates using proteomics-based strategies, including targeting, selection strategies and the application of mass spectrometry (MS) technologies and their potential impact to the neurotrauma field.

Summary

Many studies use TBI marker candidates based on literature reports, yet progress in genomics and proteomics have started to provide neurotrauma protein profiles. Choosing meaningful marker candidates from such ‘long lists’ is still pending, as only few can be taken through the process of preclinical verification and large scale translational validation. Quantitative mass spectrometry targeting specific molecules rather than random sampling of the whole proteome, e.g., multiple reaction monitoring (MRM), offers an efficient and effective means to multiplex the measurement of several candidates in patient samples, thereby omitting the need for antibodies prior to clinical assay design. Sample preparation challenges specific to TBI are addressed. A tailored selection strategy combined with a multiplex screening approach is helping to arrive at diagnostically suitable candidates for clinical assay development. A surrogate marker test will be instrumental for critical decisions of TBI patient care and protection of concussion victims from repeated exposures that could result in lasting neurological deficits.