Acute gastrointestinal permeability after traumatic brain injury in mice precedes a bloom in Akkermansia muciniphila supported by intestinal hypoxia.

Traumatic brain injury (TBI) increases gastrointestinal morbidity and associated mortality. Clinical and preclinical studies implicate gut dysbiosis as a consequence of TBI and an amplifier of brain damage. However, little is known about the association of gut dysbiosis with structural and functional changes of the gastrointestinal tract after an isolated TBI. To assess gastrointestinal dysfunction, mice received a controlled cortical impact or sham brain injury and intestinal permeability was assessed at 4 h, 8 h, 1 d, and 3 d after injury by oral administration of 4 kDa FITC Dextran prior to euthanasia. Quantification of serum fluorescence revealed an acute, short-lived increase in permeability 4 h after TBI. Despite transient intestinal dysfunction, no overt morphological changes were evident in the ileum or colon across timepoints from 4 h to 4 wks post-injury. To elucidate the timeline of microbiome changes after TBI, 16 s gene sequencing was performed on DNA extracted from fecal samples collected prior to and over the first month after TBI. Differential abundance analysis revealed that the phylum Verrucomicrobiota was increased at 1, 2, and 3 d after TBI. The Verrucomicrobiota species was identified by qPCR as Akkermansia muciniphila, an obligate anaerobe that resides in the intestinal mucus bilayer and produces short chain fatty acids (e.g. butyrate) utilized by intestinal epithelial cells. We postulated that TBI promotes intestinal changes favorable for the bloom of A. muciniphila. Consistent with this premise, the relative area of mucus-producing goblet cells in the medial colon was significantly increased at 1 d after injury, while colon hypoxia was significantly increased at 3 d. Our findings reveal acute gastrointestinal functional changes coupled with an increase of beneficial bacteria suggesting a potential compensatory response to systemic stress after TBI.

Depth-sensitive diffuse speckle contrast topography for high-density mapping of cerebral blood flow in rodents.

Significance: Frequent assessment of cerebral blood flow (CBF) is crucial for the diagnosis and management of cerebral vascular diseases. In contrast to large and expensive imaging modalities, such as nuclear medicine and magnetic resonance imaging, optical imaging techniques are portable and inexpensive tools for continuous measurements of cerebral hemodynamics. The recent development of an innovative noncontact speckle contrast diffuse correlation tomography (scDCT) enables three-dimensional (3D) imaging of CBF distributions. However, scDCT requires complex and time-consuming 3D reconstruction, which limits its ability to achieve high spatial resolution without sacrificing temporal resolution and computational efficiency. Aim: We investigate a new diffuse speckle contrast topography (DSCT) method with parallel computation for analyzing scDCT data to achieve fast and high-density two-dimensional (2D) mapping of CBF distributions at different depths without the need for 3D reconstruction. Approach: A new moving window method was adapted to improve the sampling rate of DSCT. A fast computation method utilizing MATLAB functions in the Image Processing Toolbox™ and Parallel Computing Toolbox™ was developed to rapidly generate high-density CBF maps. The new DSCT method was tested for spatial resolution and depth sensitivity in head-simulating layered phantoms and in-vivo rodent models. Results: DSCT enables 2D mapping of the particle flow in the phantom at different depths through the top layer with varied thicknesses. Both DSCT and scDCT enable the detection of global and regional CBF changes in deep brains of adult rats. However, DSCT achieves fast and high-density 2D mapping of CBF distributions at different depths without the need for complex and time-consuming 3D reconstruction. Conclusions: The depth-sensitive DSCT method has the potential to be used as a noninvasive, noncontact, fast, high resolution, portable, and inexpensive brain imager for basic neuroscience research in small animal models and for translational studies in human neonates.

The endogenous calpain inhibitor calpastatin attenuates axon degeneration in murine Guillain-Barré syndrome.

Axon degeneration accounts for the poor clinical outcome in Guillain-Barré syndrome (GBS), yet no treatments target this key pathogenic stage. Animal models demonstrate anti-ganglioside antibodies (AGAb) induce axolemmal complement pore formation through which calcium flux activates the intra-axonal calcium-dependent proteases, calpains. We previously showed protection of axonal components using soluble calpain inhibitors in ex vivo GBS mouse models, and herein, we assess the potential of axonally-restricted calpain inhibition as a neuroprotective therapy operating in vivo. Using transgenic mice that over-express the endogenous human calpain inhibitor calpastatin (hCAST) neuronally, we assessed distal motor nerve integrity in our established GBS models. We induced immune-mediated injury with monoclonal AGAb plus a source of human complement. The calpain substrates neurofilament and AnkyrinG, nerve structural proteins, were assessed by immunolabelling and in the case of neurofilament, by single-molecule arrays (Simoa). As the distal intramuscular portion of the phrenic nerve is prominently targeted in our in vivo model, respiratory function was assessed by whole-body plethysmography as the functional output in the acute and extended models. hCAST expression protects distal nerve structural integrity both ex and in vivo, as shown by attenuation of neurofilament breakdown by immunolabelling and Simoa. In an extended in vivo model, while mice still initially undergo respiratory distress owing to acute conduction failure, the recovery phase was accelerated by hCAST expression. Axonal calpain inhibition can protect the axonal integrity of the nerve in an in vivo GBS paradigm and hasten recovery. These studies reinforce the strong justification for developing further animal and human clinical studies using exogenous calpain inhibitors.

EcoHIV-Infected Mice Show No Signs of Platelet Activation.

Platelets express several surface receptors that could interact with different viruses. To understand the mechanisms of HIV-1's interaction with platelets, we chose the EcoHIV model. While EcoHIV is an established model for neuroAIDS, its effects on platelets are ill-defined. Our results indicate that EcoHIV behaves differently from HIV-1 and is cleared from circulation after 48 h post-infection. The EcoHIV course of infection resembles an HIV-1 infection under the effects of combined antiretroviral therapy (cART) since infected mice stayed immunocompetent and the virus was readily detected in the spleen. EcoHIV-infected mice failed to become thrombocytopenic and showed no signs of platelet activation. One explanation is that mouse platelets lack the EcoHIV receptor, murine Cationic Amino acid Transporter-1 (mCAT-1). No mCAT-1 was detected on their surface, nor was any mCAT-1 mRNA detected. Thus, mouse platelets would not bind or become activated by EcoHIV. However, impure virus preparations, generated by Polyethylene Glycol (PEG) precipitation, do activate platelets, suggesting that nonspecific PEG-precipitates may contain other platelet activators (e.g., histones and cell debris). Our data do not support the concept that platelets, through general surface proteins such as DC-SIGN or CLEC-2, have a wide recognition for different viruses and suggest that direct platelet/pathogen interactions are receptor/ligand specific.

Peer-induced cocaine seeking in rats: Comparison to nonsocial stimuli and role of paraventricular hypothalamic oxytocin neurons.

The purpose of this study was to determine if social vs nonsocial cues (peer vs light/tone) can serve as discriminative stimuli to reinstate cocaine seeking. In addition, to assess a potential mechanism, an oxytocin (OT) promoter-linked hM3Dq DREADD was infused into the paraventricular nucleus of the hypothalamus to determine whether peer-induced cocaine seeking is decreased by activation of OT neurons. Male rats underwent twice-daily self-administration sessions, once with cocaine in the presence of one peer (S+) and once with saline in the presence of a different peer (S-). Another experiment used similar procedures, except the discriminative stimuli were nonsocial (constant vs flashing light/tone), with one stimulus paired with cocaine (S+) and the other paired with saline (S-). A third experiment injected male and female rats with OTp-hM3Dq DREADD or control virus into PVN and tested them for peer-induced reinstatement of cocaine seeking following clozapine (0.1 mg/kg). Although acquisition of cocaine self-administration was similar in rats trained with either peer or light/tone discriminative stimuli, the latency to first response was reduced by the peer S+, but not by the light/tone S+. In addition, the effect of the conditioned stimulus was overshadowed by the peer S+ but not by the light/tone S+. Clozapine blocked the effect of the peer S+ in rats receiving the OTp-hM3Dq DREADD virus, but not in rats receiving the control virus. These results demonstrate that a social peer can serve as potent trigger for drug seeking and that OT in PVN modulates peer-induced reinstatement of cocaine seeking.

Tissue factor release following traumatic brain injury drives thrombin generation.

Background: Traumatic brain injury (TBI) results in neurovascular damage that initiates intrinsic mechanisms of hypercoagulation, which can contribute to the development of life-threatening complications, such as coagulopathy and delayed thrombosis. Clinical studies have hypothesized that tissue factor (TF) induces hypercoagulability after TBI; however, none have directly shown this relationship. Objectives: In the current study, we took a stepwise approach to understand what factors are driving thrombin generation following experimental TBI. Methods: We employed the contusion-producing controlled cortical impact (CCI) model and the diffuse closed head injury (CHI) model to investigate these mechanisms as a function of injury severity and modality. Whole blood was collected at 6 hours and 24 hours after injury, and platelet-poor plasma was used to measure thrombin generation and extracellular vesicle (EV) TF. Results: We found that plasma thrombin generation, dependent on TF present in the plasma, was greater in CCI-injured animals compared to sham at both 6 hours (120.4 ± 36.9 vs 0.0 ± 0.0 nM*min endogenous thrombin potential) and 24 hours (131.0 ± 34.0 vs 32.1 ± 20.6 nM*min) after injury. This was accompanied by a significant increase in EV TF at 24 hours (328.6 ± 62.1 vs 167.7 ± 20.8 fM) after CCI. Further, EV TF is also increased at 6 hours (126.6 ± 17.1 vs 63.3 ± 14.4 fM) but not 24 hours following CHI. Conclusion: TF-mediated thrombin generation is time-dependent after injury and TF increases resolve earlier following CHI as compared to CCI. Taken together, these data support a TF-mediated pathway of thrombin generation after TBI and pinpoint TF as a major player in TBI-induced coagulopathy.

A role for insulin-like growth factor-1 in hippocampal plasticity following traumatic brain injury.

Traumatic brain injury (TBI) initiates a constellation of secondary injury cascades, leading to neuronal damage and dysfunction that is often beyond the scope of endogenous repair mechanisms. Cognitive deficits are among the most persistent morbidities resulting from TBI, necessitating a greater understanding of mechanisms of posttraumatic hippocampal damage and neuroplasticity and identification of therapies that improve recovery by enhancing repair pathways. Focusing here on hippocampal neuropathology associated with contusion-type TBIs, the impact of brain trauma on synaptic structure and function and the process of adult neurogenesis is discussed, reviewing initial patterns of damage as well as evidence for spontaneous recovery. A case is made that insulin-like growth factor-1 (IGF-1), a growth-promoting peptide synthesized in both the brain and the periphery, is well suited to augment neuroplasticity in the injured brain. Essential during brain development, multiple lines of evidence delineate roles in the adult brain for IGF-1 in the maintenance of synapses, regulation of neurotransmission, and modulation of forms of synaptic plasticity such as long-term potentiation. Further, IGF-1 enhances adult hippocampal neurogenesis though effects on proliferation and neuronal differentiation of neural progenitor cells and on dendritic growth of newly born neurons. Post-injury administration of IGF-1 has been effective in rodent models of TBI in improving learning and memory, attenuating death of mature hippocampal neurons and promoting neurogenesis, providing critical proof-of-concept data. More studies are needed to explore the effects of IGF-1-based therapies on synaptogenesis and synaptic plasticity following TBI and to optimize strategies in order to stimulate only appropriate, functional neuroplasticity.

IGF1-Stimulated Posttraumatic Hippocampal Remodeling Is Not Dependent on mTOR.

Adult hippocampal neurogenesis is stimulated acutely following traumatic brain injury (TBI). However, many hippocampal neurons born after injury develop abnormally and the number that survive long-term is debated. In experimental TBI, insulin-like growth factor-1 (IGF1) promotes hippocampal neuronal differentiation, improves immature neuron dendritic arbor morphology, increases long-term survival of neurons born after TBI, and improves cognitive function. One potential downstream mediator of the neurogenic effects of IGF1 is mammalian target of rapamycin (mTOR), which regulates proliferation as well as axonal and dendritic growth in the CNS. Excessive mTOR activation is posited to contribute to aberrant plasticity related to posttraumatic epilepsy, spurring preclinical studies of mTOR inhibitors as therapeutics for TBI. The degree to which pro-neurogenic effects of IGF1 depend upon upregulation of mTOR activity is currently unknown. Using immunostaining for phosphorylated ribosomal protein S6, a commonly used surrogate for mTOR activation, we show that controlled cortical impact TBI triggers mTOR activation in the dentate gyrus in a time-, region-, and injury severity-dependent manner. Posttraumatic mTOR activation in the granule cell layer (GCL) and dentate hilus was amplified in mice with conditional overexpression of IGF1. In contrast, delayed astrocytic activation of mTOR signaling within the dentate gyrus molecular layer, closely associated with proliferation, was not affected by IGF1 overexpression. To determine whether mTOR activation is necessary for IGF1-mediated stimulation of posttraumatic hippocampal neurogenesis, wildtype and IGF1 transgenic mice received the mTOR inhibitor rapamycin daily beginning at 3 days after TBI, following pulse labeling with bromodeoxyuridine. Compared to wildtype mice, IGF1 overexpressing mice exhibited increased posttraumatic neurogenesis, with a higher density of posttrauma-born GCL neurons at 10 days after injury. Inhibition of mTOR did not abrogate IGF1-stimulated enhancement of posttraumatic neurogenesis. Rather, rapamycin treatment in IGF1 transgenic mice, but not in WT mice, increased numbers of cells labeled with BrdU at 3 days after injury that survived to 10 days, and enhanced the proportion of posttrauma-born cells that differentiated into neurons. Because beneficial effects of IGF1 on hippocampal neurogenesis were maintained or even enhanced with delayed inhibition of mTOR, combination therapy approaches may hold promise for TBI.

Differential Leukocyte and Platelet Profiles in Distinct Models of Traumatic Brain Injury.

Traumatic brain injury (TBI) affects over 3 million individuals every year in the U.S. There is growing appreciation that TBI can produce systemic modifications, which are in part propagated through blood-brain barrier (BBB) dysfunction and blood-brain cell interactions. As such, platelets and leukocytes contribute to mechanisms of thromboinflammation after TBI. While these mechanisms have been investigated in experimental models of contusion brain injury, less is known regarding acute alterations following mild closed head injury. To investigate the role of platelet dynamics and bioenergetics after TBI, we employed two distinct, well-established models of TBI in mice: the controlled cortical impact (CCI) model of contusion brain injury and the closed head injury (CHI) model of mild diffuse brain injury. Hematology parameters, platelet-neutrophil aggregation, and platelet respirometry were assessed acutely after injury. CCI resulted in an early drop in blood leukocyte counts, while CHI increased blood leukocyte counts early after injury. Platelet-neutrophil aggregation was altered acutely after CCI compared to sham. Furthermore, platelet bioenergetic coupling efficiency was transiently reduced at 6 h and increased at 24 h post-CCI. After CHI, oxidative phosphorylation in intact platelets was reduced at 6 h and increased at 24 h compared to sham. Taken together, these data demonstrate that brain trauma initiates alterations in platelet-leukocyte dynamics and platelet metabolism, which may be time- and injury-dependent, providing evidence that platelets carry a peripheral signature of brain injury. The unique trend of platelet bioenergetics after two distinct types of TBI suggests the potential for utilization in prognosis.

Speckle contrast diffuse correlation tomography of cerebral blood flow in perinatal disease model of neonatal piglets.

We adapted and tested an innovative noncontact speckle contrast diffuse correlation tomography (scDCT) system for 3D imaging of cerebral blood flow (CBF) variations in perinatal disease models utilizing neonatal piglets, which closely resemble human neonates. CBF variations were concurrently measured by the scDCT and an established diffuse correlation spectroscopy (DCS) during global ischemia, intraventricular hemorrhage, and asphyxia; significant correlations were observed. Moreover, CBF variations associated reasonably with vital pathophysiological changes. In contrast to DCS measurements of mixed signals from local scalp, skull and brain, scDCT generates 3D images of CBF distributions at prescribed depths within the head, thus enabling specific determination of regional cerebral ischemia. With further optimization and validation in animals and human neonates, scDCT has the potential to be a noninvasive imaging tool for both basic neuroscience research in laboratories and clinical applications in neonatal intensive care units.

Calpastatin Overexpression Protects against Excitotoxic Hippocampal Injury and Traumatic Spinal Cord Injury.

Small molecule inhibitors of calcium-dependent proteases, calpains (CAPNs), protect against neurodegeneration induced by a variety of insults including excitotoxicity and spinal cord injury (SCI). Many of these compounds, however, also inhibit other proteases, which has made it difficult to evaluate the contribution of calpains to neurodegeneration. Calpastatin is a highly specific endogenous inhibitor of classical calpains, including CAPN1 and CAPN2. In the present study, we utilized transgenic mice that overexpress human calpastatin under the prion promoter (PrP-hCAST) to evaluate the hypothesis that calpastatin overexpression protects against excitotoxic hippocampal injury and contusive SCI. The PrP-hCAST organotypic hippocampal slice cultures showed reduced neuronal death and reduced calpain-dependent proteolysis (α-spectrin breakdown production, 145 kDa) at 24 h after N-methyl-D-aspartate (NMDA) injury compared with the wild-type (WT) cultures (n = 5, p < 0.05). The PrP-hCAST mice (n = 13) displayed a significant improvement in locomotor function at one and three weeks after contusive SCI compared with the WT controls (n = 9, p < 0.05). Histological assessment of lesion volume and tissue sparing, performed on the same animals used for behavioral analysis, revealed that calpastatin overexpression resulted in a 30% decrease in lesion volume (p < 0.05) and significant increases in tissue sparing, white matter sparing, and gray matter sparing at four weeks post-injury compared with WT animals. Calpastatin overexpression reduced α-spectrin breakdown by 51% at 24 h post-injury, compared with WT controls (p < 0.05, n = 3/group). These results provide support for the hypothesis that sustained calpain-dependent proteolysis contributes to pathological deficits after excitotoxic injury and traumatic SCI.

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

Insulin-like growth factor-1 overexpression increases long-term survival of posttrauma-born hippocampal neurons while inhibiting ectopic migration following traumatic brain injury.

Cellular damage associated with traumatic brain injury (TBI) manifests in motor and cognitive dysfunction following injury. Experimental models of TBI reveal cell death in the granule cell layer (GCL) of the hippocampal dentate gyrus acutely after injury. Adult-born neurons residing in the neurogenic niche of the GCL, the subgranular zone, are particularly vulnerable. Injury-induced proliferation of neural progenitors in the subgranular zone supports recovery of the immature neuron population, but their development and localization may be altered, potentially affecting long-term survival. Here we show that increasing hippocampal levels of insulin-like growth factor-1 (IGF1) is sufficient to promote end-stage maturity of posttrauma-born neurons and improve cognition following TBI. Mice with conditional overexpression of astrocyte-specific IGF1 and wild-type mice received controlled cortical impact or sham injury and bromo-2'-deoxyuridine injections for 7d after injury to label proliferating cells. IGF1 overexpression increased the number of GCL neurons born acutely after trauma that survived 6 weeks to maturity (NeuN+BrdU+), and enhanced their outward migration into the GCL while significantly reducing the proportion localized ectopically to the hilus and molecular layer. IGF1 selectively affected neurons, without increasing the persistence of posttrauma-proliferated glia in the dentate gyrus. IGF1 overexpressing animals performed better during radial arm water maze reversal testing, a neurogenesis-dependent cognitive test. These findings demonstrate the ability of IGF1 to promote the long-term survival and appropriate localization of granule neurons born acutely after a TBI, and suggest these new neurons contribute to improved cognitive function.

Therapeutic potential of natural compounds from Chinese medicine in acute and subacute phases of ischemic stroke.

Stroke is one of the leading causes of death and disability in adults worldwide, resulting in huge social and financial burdens. Extracts from herbs, especially those used in Chinese medicine, have emerged as new pharmaceuticals for stroke treatment. Here we review the evidence from preclinical studies investigating neuroprotective properties of Chinese medicinal compounds through their application in acute and subacute phases of ischemic stroke, and highlight potential mechanisms underlying their therapeutic effects. It is noteworthy that many herbal compounds have been shown to target multiple mechanisms and in combinations may exert synergistic effects on signaling pathways, thereby attenuating multiple aspects of ischemic pathology. We conclude the paper with a general discussion of the prospects for novel natural compound-based regimens against stroke.

Fractionated mitochondrial magnetic separation for isolation of synaptic mitochondria from brain tissue.

While mitochondria maintain essential cellular functions, such as energy production, calcium homeostasis, and regulating programmed cellular death, they also play a major role in pathophysiology of many neurological disorders. Furthermore, several neurodegenerative diseases are closely linked with synaptic damage and synaptic mitochondrial dysfunction. Unfortunately, the ability to assess mitochondrial dysfunction and the efficacy of mitochondrial-targeted therapies in experimental models of neurodegenerative disease and CNS injury is limited by current mitochondrial isolation techniques. Density gradient ultracentrifugation (UC) is currently the only technique that can separate synaptic and non-synaptic mitochondrial sub-populations, though small brain regions cannot be assayed due to low mitochondrial yield. To address this limitation, we used fractionated mitochondrial magnetic separation (FMMS), employing magnetic anti-Tom22 antibodies, to develop a novel strategy for isolation of functional synaptic and non-synaptic mitochondria from mouse cortex and hippocampus without the usage of UC. We compared the yield and functionality of mitochondria derived using FMMS to those derived by UC. FMMS produced 3x more synaptic mitochondrial protein yield compared to UC from the same amount of tissue, a mouse hippocampus. FMMS also has increased sensitivity, compared to UC separation, to measure decreased mitochondrial respiration, demonstrated in a paradigm of mild closed head injury. Taken together, FMMS enables improved brain-derived mitochondrial yield for mitochondrial assessments and better detection of mitochondrial impairment in CNS injury and neurodegenerative disease.

Noninvasive noncontact speckle contrast diffuse correlation tomography of cerebral blood flow in rats.

Continuous and longitudinal imaging of cerebral blood flow (CBF) variations provide vital information to investigate pathophysiology and interventions for a variety of neurological and cerebral diseases. An innovative noncontact speckle contrast diffuse correlation tomography (scDCT) system was downscaled and adapted for noninvasive imaging of CBF distributions in rat brain through intact scalp and skull. Algorithms for 2D mapping and 3D image reconstruction of CBF distributions were developed and optimized. The continuous imaging capability of the system was shown by imaging global CBF increases during CO2 inhalations and regional CBF decreases across two hemispheres during sequential unilateral and bilateral common carotid artery ligations. The longitudinal imaging capability was demonstrated by imaging CBF variations over a long recovery period of 14 days after an acute stroke. Compared to the 2D mapping method, the 3D imaging method reduces partial volume effects, but needs more computation time for image reconstruction. Results from this study generally agree with those reported in the literature using similar protocols to induce CBF changes in rats. The scDCT enables a relatively large penetration depth (up to ∼10 mm), which is sufficient for transcranial brain measurements in small animals and human neonates. Ultimately, we expect to provide a noninvasive noncontact cerebral imager for basic neuroscience research in small animal models and clinical applications in human neonates.

Experimental Designs for Repeated Mild Traumatic Brain Injury: Challenges and Considerations.

Mild traumatic brain injury (mild TBI) is a growing public concern, as evidence mounts that even brain injuries classified as "mild" can result in persistent neurological dysfunction. Multiple brain injuries heighten the likelihood of worsened or more prolonged symptomatology and may trigger long-term neurodegeneration. Animal models provide a logical platform to identify key parameters, such as loading forces, duration between injuries, and number of injuries, which contribute to additive or synergistic damage after repeated mild TBI. Despite the tremendous increase in research productivity in the field of repeated mild TBI, relatively few studies have been designed in such a way as to provide experimental-based insights into the dependence of cellular and functional outcomes on the prescribed parameters of mild TBI. In this review, we summarize how standard models of TBI have been adapted to produce mild TBI and highlight commonly observed aspects of neuropathology replicated in rodent models of mild TBI. The complexity of designing studies of repeated TBI is discussed, including challenges of incorporating appropriate control groups, informative experimental design, and relevant outcome measures. We then feature studies that provide a well-controlled, within-study design varying either the number of injuries or the interinjury interval. Harnessing the power of experimental models of TBI to elucidate which injury parameters are critical contributors to acute and chronic damage after repeated injury can further efforts at prevention and provide improved models for testing mechanisms and therapeutic interventions.

Acute Mitochondrial Impairment Underlies Prolonged Cellular Dysfunction after Repeated Mild Traumatic Brain Injuries.

Mild traumatic brain injuries (mTBI), accounting for more than 80% of TBIs, can cause cognitive and behavioral impairments, the severity and duration of which increase after additional mTBIs. While mTBI does not cause widespread neuronal death, the mechanisms underlying increased cellular susceptibility to subsequent head impacts remain unknown. To investigate the hypothesis that altered mitochondrial bioenergetics underlie cellular vulnerability to repeated insults, we employed a mouse model of mild closed head injury (CHI) to examine mitochondrial function and oxidative stress, because these mechanisms are often intertwined. Mitochondrial respiration was assayed (Seahorse XFe24 Flux Analyzer) from cortex and hippocampus collected at 6 h, 24 h, 48 h, and 96 h post-injury. State III (adenosine diphosphate [ADP]-mediated) respiration was significantly decreased in the hippocampal mitochondria of the CHI group compared with sham at 48 h post-injury. Further, cortex-derived mitochondria exhibited a decrease in State III respiration at 24 h and 48 h post-injury. No significant differences were observed at 6 h or 96 h post-injury in either region of interest. A second CHI repeated either 48 h or 96 h after the first did not worsen State III respiration at 48 h after the final injury compared with a single CHI, but CHI repeated at a 48 h interval prolonged cortical mitochondrial dysfunction to 96 h after the final injury. Markers of oxidative stress were significantly elevated after two CHIs delivered 48 h apart, but not after single CHI or two CHI delivered 96 h apart. This study establishes that mTBI results in early mitochondrial dysfunction, which may be a determinant for cellular vulnerability to repeated head impacts. Thus, therapies targeting mitochondrial impairment could improve outcomes after repeated mTBI.

Doxorubicin-induced elevated oxidative stress and neurochemical alterations in brain and cognitive decline: protection by MESNA and insights into mechanisms of chemotherapy-induced cognitive impairment ("chemobrain").

Chemotherapy-induced cognitive impairment (CICI) is now widely recognized as a real and too common complication of cancer chemotherapy experienced by an ever-growing number of cancer survivors. Previously, we reported that doxorubicin (Dox), a prototypical reactive oxygen species (ROS)-producing anti-cancer drug, results in oxidation of plasma proteins, including apolipoprotein A-I (ApoA-I) leading to tumor necrosis factor-alpha (TNF-α)-mediated oxidative stress in plasma and brain. We also reported that co-administration of the antioxidant drug, 2-mercaptoethane sulfonate sodium (MESNA), prevents Dox-induced protein oxidation and subsequent TNF-α elevation in plasma. In this study, we measured oxidative stress in both brain and plasma of Dox-treated mice both with and without MESNA. MESNA ameliorated Dox-induced oxidative protein damage in plasma, confirming our prior studies, and in a new finding led to decreased oxidative stress in brain. This study also provides further functional and biochemical evidence of the mechanisms of CICI. Using novel object recognition (NOR), we demonstrated the Dox administration resulted in memory deficits, an effect that was rescued by MESNA. Using hydrogen magnetic resonance imaging spectroscopy (H1-MRS) techniques, we demonstrated that Dox administration led to a dramatic decrease in choline-containing compounds assessed by (Cho)/creatine ratios in the hippocampus in mice. To better elucidate a potential mechanism for this MRS observation, we tested the activities of the phospholipase enzymes known to act on phosphatidylcholine (PtdCho), a key component of phospholipid membranes and a source of choline for the neurotransmitter, acetylcholine (ACh). The activities of both phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D were severely diminished following Dox administration. The activity of PC-PLC was preserved when MESNA was co-administered with Dox; however, PLD activity was not protected. This study is the first to demonstrate the protective effects of MESNA on Dox-related protein oxidation, cognitive decline, phosphocholine (PCho) levels, and PC-PLC activity in brain and suggests novel potential therapeutic targets and strategies to mitigate CICI.

Assessment of systemic administration of PEGylated IGF-1 in a mouse model of traumatic brain injury.

BACKGROUND: Traumatic brain injury can result in lasting cognitive dysfunction due to degeneration of mature hippocampal neurons as well as the loss of immature neurons within the dentate gyrus. While endogenous neurogenesis affords a partial recovery of the immature neuron population, hippocampal neurogenesis may be enhanced through therapeutic intervention. Insulin-like growth factor-1 (IGF-1) has the potential to improve cognitive function and promote neurogenesis after TBI, but its short half-life in the systemic circulation makes it difficult to maintain a therapeutic concentration. IGF-1 modified with a polyethylene glycol moiety (PEG-IGF-1) exhibits improved stability and half-life while retaining its ability to enter the brain from the periphery, increasing its viability as a translational approach. OBJECTIVE: The goal of this study was to evaluate the ability of systemic PEG-IGF-1 administration to attenuate acute neuronal loss and stimulate the recovery of hippocampal immature neurons in brain-injured mice. METHODS: In a series of studies utilizing a well-established contusion brain injury model, PEG-IGF-1 was administered subcutaneously after injury. Serum levels of PEG were verified using ELISA and histological staining was used to investigate numbers of degenerating neurons and cortical contusion size at 24 h after injury. Immunofluorescent staining was used to evaluate numbers of immature neurons at 10 d after injury. RESULTS: Although subcutaneous injections of PEG-IGF-1 increased serum IGF-1 levels in a dose-dependent manner, no effects were observed on cortical contusion size, neurodegeneration within the dentate gyrus, or recovery of hippocampal immature neuron numbers. CONCLUSIONS: In contrast to its efficacy in rodent models of neurodegenerative diseases, PEG- IGF-1 was not effective in ameliorating early neuronal loss after contusion brain trauma.