The evolution of traumatic brain injury in a rat focal contusion model.

Serial MRI facilitates the in vivo analysis of the intra- and intersubject evolution of traumatic brain injury lesions. Despite the availability of MRI, the natural history of experimental focal contusion lesions in the controlled cortical impact (CCI) rat model has not been well described. We performed CCI on rats and MRI during the acute to chronic stages of cerebral injury to investigate the time course of changes in the brain. Female Wistar rats underwent CCI of their left motor cortex with a flat impact tip driven by an electromagnetic piston. In vivo MRI was performed at 7 T serially over 6 weeks post-CCI. The appearances of CCI-induced lesions and lesion-associated cortical volumes were variable on MRI, with the percentage change in cortical volume of the CCI ipsilateral side relative to the contralateral side ranging from 18% within 2 h of injury on day 0 to a peak of 35% on day 1, and a trough of -28% by week 5/6, with an average standard deviation of ± 14% at any given time point. In contrast, the percentage change in cortical volume of the ipsilateral side relative to the contralateral side in control rats was not significant (1 ± 2%). Hemorrhagic conversion within and surrounding the CCI lesion occurred between days 2 and 9 in 45% of rats, with no hemorrhage noted on the initial scan. Furthermore, hemorrhage and hemosiderin within the lesion were positive for Prussian blue and highly autofluorescent on histological examination. Although some variation in injuries may be technique related, the divergence of similar lesions between initial and final scans demonstrates the inherent biological variability of the CCI rat model.

Enhanced homing permeability and retention of bone marrow stromal cells by noninvasive pulsed focused ultrasound.

Bone marrow stromal cells (BMSCs) have shown significant promise in the treatment of disease, but their therapeutic efficacy is often limited by inefficient homing of systemically administered cells, which results in low number of cells accumulating at sites of pathology. BMSC home to areas of inflammation where local expression of integrins and chemokine gradients is present. We demonstrated that nondestructive pulsed focused ultrasound (pFUS) exposures that emphasize the mechanical effects of ultrasound-tissue interactions induced local and transient elevations of chemoattractants (i.e., cytokines, integrins, and growth factors) in the murine kidney. pFUS-induced upregulation of cytokines occurred through approximately 1 day post-treatment and returned to contralateral kidney levels by day 3. This window of significant increases in cytokine expression was accompanied by local increases of other trophic factors and integrins that have been shown to promote BMSC homing. When BMSCs were intravenously administered following pFUS treatment to a single kidney, enhanced homing, permeability, and retention of BMSC was observed in the treated kidney versus the contralateral kidney. Histological analysis revealed up to eight times more BMSC in the peritubular regions of the treated kidneys on days 1 and 3 post-treatment. Furthermore, cytokine levels in pFUS-treated kidneys following BMSC administration were found to be similar to controls, suggesting modulation of cytokine levels by BMSC. pFUS could potentially improve cell-based therapies as a noninvasive modality to target homing by establishing local chemoattractant gradients and increasing expression of integrins to enhance tropism of cells toward treated tissues.

Phase contrast MRI is an early marker of micrometastatic breast cancer development in the rat brain.

The early growth of micrometastatic breast cancer in the brain often occurs through vessel co-option and is independent of angiogenesis. Remodeling of the existing vasculature is an important step in the evolution of co-opting micrometastases into angiogenesis-dependent solid tumor masses. The purpose of this study was to determine whether phase contrast MRI, an intrinsic source of contrast exquisitely sensitive to the magnetic susceptibility properties of deoxygenated hemoglobin, could detect vascular changes occurring independent of angiogenesis in a rat model of breast cancer metastases to the brain. Twelve nude rats were administered 10(6) MDA-MB-231BRL 'brain-seeking' breast cancer cells through intracardiac injection. Serial, multiparametric MRI of the brain was performed weekly until metastatic disease was detected. The results demonstrated that images of the signal phase (area under the receiver operating characteristic curve, 0.97) were more sensitive than T(2)* gradient echo magnitude images (area under the receiver operating characteristic curve, 0.73) to metastatic brain lesions. The difference between the two techniques was probably the result of the confounding effects of edema on the magnitude of the signal. A region of interest analysis revealed that vascular abnormalities detected with phase contrast MRI preceded tumor permeability measured with contrast-enhanced MRI by 1-2 weeks. Tumor size was correlated with permeability (R(2)= 0.23, p < 0.01), but phase contrast was independent of tumor size (R(2)= 0.03). Histopathologic analysis demonstrated that capillary endothelial cells co-opted by tumor cells were significantly enlarged, but less dense, relative to the normal brain vasculature. Although co-opted vessels were vascular endothelial growth factor-negative, vessels within larger tumor masses were vascular endothelial growth factor-positive. In conclusion, phase contrast MRI is believed to be sensitive to vascular remodeling in co-opting brain tumor metastases independent of sprouting angiogenesis, and may therefore aid in preclinical studies of angiogenic-independent tumors or in the monitoring of continued tumor growth following anti-angiogenic therapy. Published 2011. This article is a US Government work and is in the public domain in the USA.

The contribution of gliosis to diffusion tensor anisotropy and tractography following traumatic brain injury: validation in the rat using Fourier analysis of stained tissue sections.

Diffusion tensor imaging is highly sensitive to the microstructural integrity of the brain and has uncovered significant abnormalities following traumatic brain injury not appreciated through other methods. It is hoped that this increased sensitivity will aid in the detection and prognostication in patients with traumatic injury. However, the pathological substrates of such changes are poorly understood. Specifically, decreases in fractional anisotropy derived from diffusion tensor imaging are consistent with axonal injury, myelin injury or both in white matter fibres. In contrast, in both humans and animal models, increases in fractional anisotropy have been suggested to reflect axonal regeneration and plasticity, but the direct histological evidence for such changes remains tenuous. We developed a method to quantify the anisotropy of stained histological sections using Fourier analysis, and applied the method to a rat controlled cortical impact model to identify the specific pathological features that give rise to the diffusion tensor imaging changes in subacute to chronic traumatic brain injury. A multiple linear regression was performed to relate the histological measurements to the measured diffusion tensor changes. The results show that anisotropy was significantly increased (P < 0.001) in the perilesioned cortex following injury. Cortical anisotropy was independently associated (standardized ß = 0.62, P = 0.04) with the coherent organization of reactive astrocytes (i.e. gliosis) and was not attributed to axons. By comparison, a decrease in white matter anisotropy (P < 0.001) was significantly related to demyelination (ß = 0.75, P = 0.0015) and to a lesser extent, axonal degeneration (ß = -0.48, P = 0.043). Gliosis within the lesioned cortex also influenced diffusion tensor tractography, highlighting the fact that spurious tracts in the injured brain may not necessarily reflect continuous axons and may instead depict glial scarring. The current study demonstrates a novel method to relate pathology to diffusion tensor imaging findings, elucidates the underlying mechanisms of anisotropy changes following traumatic brain injury and significantly impacts the clinical interpretation of diffusion tensor imaging findings in the injured brain.

Quantitative T2* imaging of metastatic human breast cancer to brain in the nude rat at 3 T.

This study uses quantitative T(2)* imaging to track ferumoxides--protamine sulfate (FEPro)-labeled MDA-MB-231BR-Luc (231BRL) human breast cancer cells that metastasize to the nude rat brain. Four cohorts of nude rats were injected intracardially with FEPro-labeled, unlabeled or tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)-treated (to induce apoptosis) 231BRL cells, or saline, in order to develop metastatic breast cancer in the brain. The heads of the rats were imaged serially over 3-4 weeks using gradient multi-echo and turbo spin-echo pulse sequences at 3 T with a solenoid receive-only 4-cm-diameter coil. Quantitative T(2)* maps of the whole brain were obtained by the application of single-exponential fitting to the signal intensity of T(2)* images, and the distribution of T(2)* values in brain voxels was calculated. MRI findings were correlated with Prussian blue staining and immunohistochemical staining for iron in breast cancer and macrophages. Quantitative analysis of T(2)* from brain voxels demonstrated a significant shift to lower values following the intracardiac injection of FEPro-labeled 231BRL cells, relative to animals receiving unlabeled cells, apoptotic cells or saline. Quartile analysis based on the T(2)* distribution obtained from brain voxels demonstrated significant differences (p < 0.0083) in the number of voxels with T(2)* values in the ranges 10-35 ms (Q1), 36-60 ms (Q2) and 61-86 ms (Q3) from 1 day to 3 weeks post-infusion of labeled 231BRL cells, compared with baseline scans. There were no significant differences in the distribution of T(2)* obtained from serial MRI in rats receiving unlabeled or TRAIL-treated cells or saline. Histologic analysis demonstrated isolated Prussian blue-positive breast cancer cells scattered in the brains of rats receiving labeled cells, relative to animals receiving unlabeled or apoptotic cells. Quantitative T(2)* analysis of FEPro-labeled metastasized cancer cells was possible even after the hypointense voxels were no longer visible on T(2)*-weighted images.

Enhanced stem cell tracking via electrostatically assembled fluorescent SPION-peptide complexes.

For cellular MRI there is a need to label cells with superparamagnetic iron oxide nanoparticles (SPION) that have multiple imaging moieties that are nontoxic and have increased NMR relaxation properties to improve the detection and tracking of therapeutic cells. Although increases in the relaxation properties of SPION have been accomplished, detection of tagged cells is limited by either poor cell labeling efficiency or low intracellular iron content. A strategy via a complex formation with transfection agents to overcome these obstacles has been reported. In this paper, we report a complex formation between negatively charged fluorescent monodisperse SPION and positively charged peptides and use the complex formation to improve the MR properties of labeled stem cells. As a result, labeled stem cells exhibited a strong fluorescent signal and enhanced T 2*-weighted MR imaging in vitro and in vivo in a flank tumor model.

Repeated Mild Closed Head Injuries Induce Long-Term White Matter Pathology and Neuronal Loss That Are Correlated With Behavioral Deficits.

An estimated 5.3 million Americans are living with a disability from a traumatic brain injury (TBI). There is emerging evidence of the detrimental effects from repeated mild TBIs (rmTBIs). rmTBI manifests its own unique set of behavioral and neuropathological changes. A subset of individuals exposed to rmTBI develop permanent behavioral and pathological consequences, defined postmortem as chronic traumatic encephalopathy. We have combined components of two classic rodent models of TBI, the controlled cortical impact model and the weight drop model, to develop a repeated mild closed head injury (rmCHI) that produces long-term deficits in several behaviors that correlate with neuropathological changes. Mice receiving rmCHI performed differently from 1-hit or sham controls on the elevated plus maze; these deficits persist up to 6 months postinjury (MPI). rmCHI mice performed worse than 1-hit and control sham mice at 2 MPI and 6 MPI on the Morris water maze. Mice receiving rmCHI exhibited significant atrophy of the corpus callosum at both 2 MPI and 6 MPI, as assessed by stereological volume analysis. Stereological analysis also revealed significant loss of cortical neurons in comparison with 1-hit and controls. Moreover, both of these pathological changes correlated with behavioral impairments. In human tau transgenic mice, rmCHI induced increases in hyperphosphorylated paired helical filament 1 tau in the hippocampus. This suggests that strategies to restore myelination or reduce neuronal loss may ameliorate the behavioral deficits observed following rmCHI and that rmCHI may model chronic traumatic encephalopathy in human tau mice.

Effects of concussion on the blood-brain barrier in humans and rodents.

Traumatic brain injury and the long-term consequences of repeated concussions constitute mounting concerns in the United States, with 5.3 million individuals living with a traumatic brain injury-related disability. Attempts to understand mechanisms and possible therapeutic approaches to alleviate the consequences of repeat mild concussions or traumatic brain injury on cerebral vasculature depend on several aspects of the trauma, including: (1) the physical characteristics of trauma or insult that result in damage; (2) the time "window" after trauma in which neuropathological features develop; (3) methods to detect possible breakdown of the blood-brain barrier; and (4) understanding different consequences of a single concussion as compared with multiple concussions. We review the literature to summarize the current understanding of blood-brain barrier and endothelial cell changes post-neurotrauma in concussions and mild traumatic brain injury. Attention is focused on concussion and traumatic brain injury in humans, with a goal of pointing out the gaps in our knowledge and how studies of rodent model systems of concussion may help in filling these gaps. Specifically, we focus on disruptions that concussion causes to the blood-brain barrier and its multifaceted consequences. Importantly, the magnitude of post-concussion blood-brain barrier dysfunction may influence the time course and extent of neuronal recovery; hence, we include in this review comparisons of more severe traumatic brain injury to concussion where appropriate. Finally, we address the important, and still unresolved, issue of how best to detect possible breakdown in the blood-brain barrier following neurotrauma by exploring intravascular tracer injection in animal models to examine leakage into the brain parenchyma.

Effects of Human ES-Derived Neural Stem Cell Transplantation and Kindling in a Rat Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) is one of the leading causes of death and disability in the population worldwide, with a broad spectrum of symptoms and disabilities. Posttraumatic hyperexcitability is one of the most common neurological disorders that affect people after a head injury. A reliable animal model of posttraumatic hyperexcitability induced by TBI which allows one to test effective treatment strategies is yet to be developed. To address these issues, in the present study, we tested human embryonic stem cell-derived neural stem cell (NSC) transplantation in an animal model of posttraumatic hyperexcitability in which the brain injury was produced in one hemisphere of immunodeficient athymic nude rats by controlled cortical impact, and spontaneous seizures were produced by repeated electrical stimulation (kindling) in the contralateral hemisphere. At 14 wk posttransplantation, we report human NSC (hNSC) survival and differentiation into all 3 neural lineages in both sham and injured animals. We observed twice as many surviving hNSCs in the injured versus sham brain, and worse survival on the kindled side in both groups, indicating that kindling/seizures are detrimental to survival or proliferation of hNSCs. We also replicated our previous finding that hNSCs can ameliorate deficits on the novel place recognition task,33 but such improvements are abolished following kindling. We found no significant differences pre- or post-kindling on the elevated plus maze. No significant correlations were observed between hNSC survival and cognitive performance on either task. Together these findings suggest that Shef6-derived hNSCs may be beneficial as a therapy for TBI, but not in animals or patients with posttraumatic hyperexcitability.

Transplantation of human neural stem cells restores cognition in an immunodeficient rodent model of traumatic brain injury.

Traumatic brain injury (TBI) in humans can result in permanent tissue damage and has been linked to cognitive impairment that lasts years beyond the initial insult. Clinically effective treatment strategies have yet to be developed. Transplantation of human neural stem cells (hNSCs) has the potential to restore cognition lost due to injury, however, the vast majority of rodent TBI/hNSC studies to date have evaluated cognition only at early time points, typically <1month post-injury and cell transplantation. Additionally, human cell engraftment and long-term survival in rodent models of TBI has been difficult to achieve due to host immunorejection of the transplanted human cells, which confounds conclusions pertaining to transplant-mediated behavioral improvement. To overcome these shortfalls, we have developed a novel TBI xenotransplantation model that utilizes immunodeficient athymic nude (ATN) rats as the host recipient for the post-TBI transplantation of human embryonic stem cell (hESC) derived NSCs and have evaluated cognition in these animals at long-term (≥2months) time points post-injury. We report that immunodeficient ATN rats demonstrate hippocampal-dependent spatial memory deficits (Novel Place, Morris Water Maze), but not non-spatial (Novel Object) or emotional/anxiety-related (Elevated Plus Maze, Conditioned Taste Aversion) deficits, at 2-3months post-TBI, confirming that ATN rats recapitulate some of the cognitive deficits found in immunosufficient animal strains. Approximately 9-25% of transplanted hNSCs survived for at least 5months post-transplantation and differentiated into mature neurons (NeuN, 18-38%), astrocytes (GFAP, 13-16%), and oligodendrocytes (Olig2, 11-13%). Furthermore, while this model of TBI (cortical impact) targets primarily cortex and the underlying hippocampus and generates a large lesion cavity, hNSC transplantation facilitated cognitive recovery without affecting either lesion volume or total spared cortical or hippocampal tissue volume. Instead, we have found an overall increase in host hippocampal neuron survival in hNSC transplanted animals and demonstrate that a correlation exists between hippocampal neuron survival and cognitive performance. Together, these findings support the use of immunodeficient rodents in models of TBI that involve the transplantation of human cells, and suggest that hNSC transplantation may be a viable, long-term therapy to restore cognition after brain injury.

Phenotypic assessment of galanin overexpressing and galanin receptor R1 knockout mice in the tail suspension test for depression-related behavior.

RATIONALE: Galanin and its receptors exert inhibitory neuromodulatory control over brain monoamines. Rat studies revealed that galanin expression is upregulated by exposure to stressors and that galanin manipulations modify neuroendocrine and behavioral responses to stress, leading to the hypothesis that galanin mediates depression-related behaviors. METHODS: In the present study, we examined the role of galanin in modulating antidepressant-related behavior in galanin overexpressing transgenic (GAL-tg) mice and galanin receptor R1 knockout (GAL-R1 KO) mice, using the tail suspension test (TST). Quantitative autoradiography for 5-HT(1A)-R and serotonin transporter binding density tested for changes in these two major regulatory components of the 5-HT system in galanin mutant mice. RESULTS: Baseline TST behavior was normal in GAL-tg and GAL-R1 KO mice, and intracerebroventricular administration of galanin failed to alter TST behavior in normal C57BL/6J mice. The TST anti-immobility effects of acute treatment with the serotonin reuptake inhibitor, fluoxetine (0-30 mg/kg), and the norepinephrine reuptake inhibitor, desipramine (0-30 mg/kg), were unaltered in galanin mutant mice. Hippocampal 5-HT(1A)-R density was significantly elevated in GAL-tg and GAL-R1 KO mice, while hippocampal 5-HTT density was reduced in GAL-R1 KO mice, relative to controls. CONCLUSION: Neither pharmacological nor molecular genetic manipulations of galanin altered depression-related profiles in the TST. Possible functional alterations in hippocampal 5-HT neurotransmission may have contributed to these negative results. These preliminary findings provide evidence against the hypothesis that galanin plays a central role in mouse depression-related behaviors. It remains possible that galanin modulates depression-related responses in other experimental paradigms and species.

Failure of intravenous or intracardiac delivery of mesenchymal stromal cells to improve outcomes after focal traumatic brain injury in the female rat.

Mesenchymal stromal cells secrete a variety of anti-inflammatory factors and may provide a regenerative medicine option for the treatment of traumatic brain injury. The present study investigates the efficacy of multiple intravenous or intracardiac administrations of rat mesenchymal stromal cells or human mesenchymal stromal cells in female rats after controlled cortical impact by in vivo MRI, neurobehavior, and histopathology evaluation. Neither intravenous nor intracardiac administration of mesenchymal stromal cells derived from either rats or humans improved MRI measures of lesion volume or neurobehavioral outcome compared to saline treatment. Few mesenchymal stromal cells (<0.0005% of injected dose) were found within 3 days of last dosage at the site of injury after either delivery route, with no mesenchymal stromal cells being detectable in brain at 30 or 56 days post-injury. These findings suggest that non-autologous mesenchymal stromal cells therapy via intravenous or intracardiac administration is not a promising treatment after focal contusion traumatic brain injury in this female rodent model.

CD133-enriched Xeno-Free human embryonic-derived neural stem cells expand rapidly in culture and do not form teratomas in immunodeficient mice.

Common methods for the generation of human embryonic-derived neural stem cells (hNSCs) result in cells with potentially compromised safety profiles due to maintenance of cells in conditions containing non-human proteins (e.g. in bovine serum or on mouse fibroblast feeders). Additionally, sufficient expansion of resulting hNSCs for scaling out or up in a clinically relevant time frame has proven to be difficult. Here, we report a strategy that produces hNSCs in completely "Xeno-Free" culture conditions. Furthermore, we have enriched the hNSCs for the cell surface marker CD133 via magnetic sorting, which has led to an increase in the expansion rate and neuronal fate specification of the hNSCs in vitro. Critically, we have also confirmed neural lineage specificity upon sorted hNSC transplantation into the immunodeficient NOD-scid mouse brain. The future use or adaptation of these protocols has the potential to better facilitate the advancement of pre-clinical strategies from the bench to the bedside.

Functional assessment of long-term deficits in rodent models of traumatic brain injury.

Traumatic brain injury (TBI) ranks as the leading cause of mortality and disability in the young population worldwide. The annual US incidence of TBI in the general population is estimated at 1.7 million per year, with an estimated financial burden in excess of US$75 billion a year in the USA alone. Despite the prevalence and cost of TBI to individuals and society, no treatments have passed clinical trial to clinical implementation. The rapid expansion of stem cell research and technology offers an alternative to traditional pharmacological approaches targeting acute neuroprotection. However, preclinical testing of these approaches depends on the selection and characterization of appropriate animal models. In this article we consider the underlying pathophysiology for the focal and diffuse TBI subtypes, discuss the existing preclinical TBI models and functional outcome tasks used for assessment of injury and recovery, identify criteria particular to preclinical animal models of TBI in which stem cell therapies can be tested for safety and efficacy, and review these criteria in the context of the existing TBI literature. We suggest that 2 months post-TBI is the minimum period needed to evaluate human cell transplant efficacy and safety. Comprehensive review of the published TBI literature revealed that only 32% of rodent TBI papers evaluated functional outcome ≥1 month post-TBI, and only 10% evaluated functional outcomes ≥2 months post-TBI. Not all published papers that evaluated functional deficits at a minimum of 2 months post-TBI reported deficits; hence, only 8.6% of overall TBI papers captured in this review demonstrated functional deficits at 2 months or more postinjury. A 2-month survival and assessment period would allow sufficient time for differentiation and integration of human neural stem cells with the host. Critically, while trophic effects might be observed at earlier time points, it will also be important to demonstrate the sustainability of such an effect, supporting the importance of an extended period of in vivo observation. Furthermore, regulatory bodies will likely require at least 6 months survival post-transplantation for assessment of toxicology/safety, particularly in the context of assessing cell abnormalities.

Differential microstructure and physiology of brain and bone metastases in a rat breast cancer model by diffusion and dynamic contrast enhanced MRI.

Pharmacological approaches to treat breast cancer metastases in the brain have been met with limited success. In part, the impermeability of the blood brain barrier (BBB) has hindered delivery of chemotherapeutic agents to metastatic tumors in the brain. BBB-permeable chemotherapeutic drugs are being developed, and noninvasively assessing the efficacy of these agents will be important in both preclinical and clinical settings. In this regard, dynamic contrast enhanced (DCE) and diffusion weighted imaging (DWI) are magnetic resonance imaging (MRI) techniques to monitor tumor vascular permeability and cellularity, respectively. In a rat model of metastatic breast cancer, we demonstrate that brain and bone metastases develop with distinct physiological characteristics as measured with MRI. Specifically, brain metastases have limited permeability of the BBB as assessed with DCE and an increased apparent diffusion coefficient (ADC) measured with DWI compared to the surrounding brain. Microscopically, brain metastases were highly infiltrative, grew through vessel co-option, and caused extensive edema and injury to the surrounding neurons and their dendrites. By comparison, metastases situated in the leptomenengies or in the bone had high vascular permeability and significantly lower ADC values suggestive of hypercellularity. On histological examination, tumors in the bone and leptomenengies were solid masses with distinct tumor margins. The different characteristics of these tissue sites highlight the influence of the microenvironment on metastatic tumor growth. In light of these results, the suitability of DWI and DCE to evaluate the response of chemotherapeutic and anti-angiogenic agents used to treat co-opted brain metastases, respectively, remains a formidable challenge.

Craniotomy: true sham for traumatic brain injury, or a sham of a sham?

Abstract Neurological dysfunction after traumatic brain injury (TBI) is caused by both the primary injury and a secondary cascade of biochemical and metabolic events. Since TBI can be caused by a variety of mechanisms, numerous models have been developed to facilitate its study. The most prevalent models are controlled cortical impact and fluid percussion injury. Both typically use "sham" (craniotomy alone) animals as controls. However, the sham operation is objectively damaging, and we hypothesized that the craniotomy itself may cause a unique brain injury distinct from the impact injury. To test this hypothesis, 38 adult female rats were assigned to one of three groups: control (anesthesia only); craniotomy performed by manual trephine; or craniotomy performed by electric dental drill. The rats were then subjected to behavioral testing, imaging analysis, and quantification of cortical concentrations of cytokines. Both craniotomy methods generate visible MRI lesions that persist for 14 days. The initial lesion generated by the drill technique is significantly larger than that generated by the trephine. Behavioral data mirrored lesion volume. For example, drill rats have significantly impaired sensory and motor responses compared to trephine or naïve rats. Finally, of the seven tested cytokines, KC-GRO and IFN-γ showed significant increases in both craniotomy models compared to naïve rats. We conclude that the traditional sham operation as a control confers profound proinflammatory, morphological, and behavioral damage, which confounds interpretation of conventional experimental brain injury models. Any experimental design incorporating "sham" procedures should distinguish among sham, experimentally injured, and healthy/naïve animals, to help reduce confounding factors.