Multi-modal approach for investigating brain and behavior changes in an animal model of traumatic brain injury

Utilization of novel approaches in imaging modalities are needed for enhancing diagnostic and therapeutic outcomes of persons suffering a traumatic brain injury (TBI). This study explored the feasibility of using functional magnetic resonance imaging (fMRI) in conjunction with behavioral measures to target dynamic changes in specific neural circuitries in an animal model of traumatic brain injury. Wistar rats were randomly assigned to one of two groups (traumatic brain injury / sham operation). TBI rats were subjected to the closed head injury (CHI) model. Any observable motor deficits and cognitive deficits associated with the injury were measured using Beam Walk and Morris Water Maze tests, respectively. fMRI was performed to assess the underlying post-traumatic cerebral anatomy and function in acute (24 hours after the injury) and chronic (7 and 21 days after the injury) phases. Beam Walk test results detected no significant differences in motor deficits between groups. Morris Water Maze test indicated that cognitive deficits persisted for the first week following injury and to a large extent, recovered thereafter. Resting state functional connectivity (rsFC) analysis detected initially diminished connectivity between cortical areas involved in cognition for the TBI group; however the connectivity patterns normalized at one week and remained so at three weeks post-injury timepoint. Taken together, we have demonstrated an objective in vivo marker for mapping functional brain changes correlated with injury-associated cognitive behavior deficits and offer an animal model for testing potential therapeutic interventions options

Animal models of focal brain ischemia

Stroke is a leading cause of disability and death in many countries. Understanding the pathophysiology of ischemic injury and developing therapies is an important endeavor that requires much additional research. Animal stroke models provide an important mechanism for these activities. A large number of stroke models have been developed and are currently used in laboratories around the world. These models are overviewed as are approaches for measuring infarct size and functional outcome

Transforming Growth Factor: β Signaling Is Essential for Limb Regeneration in Axolotls

Axolotls (urodele amphibians) have the unique ability, among vertebrates, to perfectly regenerate many parts of their body including limbs, tail, jaw and spinal cord following injury or amputation. The axolotl limb is the most widely used structure as an experimental model to study tissue regeneration. The process is well characterized, requiring multiple cellular and molecular mechanisms. The preparation phase represents the first part of the regeneration process which includes wound healing, cellular migration, dedifferentiation and proliferation. The redevelopment phase represents the second part when dedifferentiated cells stop proliferating and redifferentiate to give rise to all missing structures. In the axolotl, when a limb is amputated, the missing or wounded part is regenerated perfectly without scar formation between the stump and the regenerated structure. Multiple authors have recently highlighted the similarities between the early phases of mammalian wound healing and urodele limb regeneration. In mammals, one very important family of growth factors implicated in the control of almost all aspects of wound healing is the transforming growth factor-beta family (TGF-β). In the present study, the full length sequence of the axolotl TGF-β1 cDNA was isolated. The spatio-temporal expression pattern of TGF-β1 in regenerating limbs shows that this gene is up-regulated during the preparation phase of regeneration. Our results also demonstrate the presence of multiple components of the TGF-β signaling machinery in axolotl cells. By using a specific pharmacological inhibitor of TGF-β type I receptor, SB-431542, we show that TGF-β signaling is required for axolotl limb regeneration. Treatment of regenerating limbs with SB-431542 reveals that cellular proliferation during limb regeneration as well as the expression of genes directly dependent on TGF-β signaling are down-regulated. These data directly implicate TGF-β signaling in the initiation and control of the regeneration process in axolotls

Multimodal MRI, Behavioral Testing, and Histology in a Rat Model of Transient Focal Cerebral Ischemia : A Dissertation

Cerebral ischemia is defined as a decrease in blood flow to the brain. It is most often caused by obstruction of a cerebral blood vessel, and is recognized by the World Health Organization as the leading cause of serious adult disability and one of the top three causes of adult death worldwide. Most survivors demonstrate partial restitution of function over time, but the underlying recovery mechanism(s) remain unclear especially in a subset of patients with persistent neurological morbidities despite normal-appearing brain on neuroimaging. The optimal way to understand any human disease state is via clinical studies. Unfortunately, well-controlled experiments in humans are difficult due to small patient populations, the presence of numerous confounding variables, and ethical issues associated with invasive or discomforting experimental procedures. Anesthetized animal models of cerebral ischemia afford a means of avoiding the above difficulties. However, anesthesia and physiological perturbations that occasionally follow brain ischemia may affect the reliability of certain tools used to study this disease, such as functional magnetic resonance imaging (fMRI). Therefore, the central goals of this thesis were: 1) to evaluate the feasibility of performing fMRI in anesthetized and awake animals, 2) to assess fMRI responses under various perturbations of cerebral perfusion and tissue oxygenation in order to identify key factors that may modulate functional signal changes following ischemia, and 3) to utilize fMRI, behavioral tests and histology in an anesthetized animal model of transient focal cerebral ischemia to explore postischemic changes in brain pathology/function and how they relate to changes in behavior. In the first study of this dissertation, I report the evaluation of fMRI responses in anesthetized and awake animals. Anesthesia is frequently used in animal models of cerebral ischemia, but is known to alter brain perfusion and metabolism which may, in turn, affect fMRI responsivity. Perfusion-based fMRI was used to evaluate cerebral blood flow (CBF) and blood oxygenation level-dependent (BOLD) responses to hypercapnia in awake and isoflurane-anesthetized rats. Hypercapnia produced significant CBF and BOLD fMRI signal changes throughout the cerebrum in awake and isoflurane-anesthetized groups. These results show that perfusion-based fMRI can successfully detect stimulus-evoked hemodynamic changes in the brains of both conscious and isoflurane-anesthetized animals. The second study of this dissertation: 1) investigates the effects of alterations in cerebral perfusion and oxygenation on fMRI signal changes, and 2) examines the self-consistency of an imaging-based formalism for the calculation of the cerebral metabolic rate of oxygen (CMRO2). Functional MRI responses to a stimulus can be described in terms of relative or absolute signal change. A relative fMRI response is defined as a percent-change relative to its own respective baseline value. An absolute fMRI response is defined as a quantitative change relative to a single fixed baseline value that serves as a control. Thus, an absolute fMRI signal change is largely independent of the baseline state and may more accurately index brain activity when baseline fMRI signals change significantly over time due to, for example, hemodynamic-metabolic disturbances that occur during and/or after brain ischemia. To address these issues, the effects of inspired hypoxic, normoxic, hyperoxic, and hypercapnic gases on baseline and forepaw stimulation-evoked changes in BOLD and CBF fMRI signals were examined in isoflurane-anesthetized rats. Relative fMRI responses to forepaw stimulation varied-whereas. absolute responses were similar--across gas conditions. These results demonstrate that absolute measurements of fMRI signal change may lend a more accurate measure of brain activity during states of altered basal physiology as well as support the self-consistency of the imaging-based CMRO2 formalism under these conditions. The third and last study of this dissertation utilized multimodal MRI, behavioral tests, and histology at acute to chronic periods following transient middle cerebral artery occlusion (tMCAO) in the rat to examine the evolution of pathological, functional, and behavioral parameters following transient focal cerebral ischemia. MRI was used to track the evolution of brain pathology and function following cerebral ischemia, and it was found that the cerebral sensorimotor network, critical for sensory and motor behavioral functions, showed profoundly abnormal signal changes that required up to one day to normalize. Adhesive removal, forepaw placement and beam-walk behavioral tests demonstrated sensorimotor dysfunctions that gradually improved but remained long after the recovery of MRI parameters. Postmortem histology confirmed the presence of selective neural cell death within the sensorimotor network at time points when behavior was abnormal. These results suggest that subtle postischemic pathological changes in the brain undetectable by MRI may be responsible for persistent behavioral deficits-a finding which may be relevant to a clinical subset of patients with persistent neurological morbidities despite negative MRI results following cerebral ischemia

Emerging drugs for acute ischemic stroke

Stroke is no longer an untreatable condition, and its management is changing rapidly as new developments appear for acute treatments. The objective of this article is to provide a concise introduction to the pathophysiology and existing treatments for stroke, as well as discussing current research goals and interventions under evaluation, along with potential development issues. It will end with the authors\u27 interpretations of the presented material and their opinions about where the field is going, or should go, in the coming years. The material herein is derived from current literature and expert opinion. Overall, the most important findings are that future advances in stroke treatment will probably include combination therapies that will be administered in specialized stroke care units to maximize patient outcome

Effects of hypoxia, hyperoxia, and hypercapnia on baseline and stimulus-evoked BOLD, CBF, and CMRO2 in spontaneously breathing animals

Functional magnetic resonance imaging (fMRI) was used to investigate the effects of inspired hypoxic, hyperoxic, and hypercapnic gases on baseline and stimulus-evoked changes in blood oxygenation level-dependent (BOLD) signals, cerebral blood flow (CBF), and the cerebral metabolic rate of oxygen (CMRO2) in spontaneously breathing rats under isoflurane anesthesia. Each animal was subjected to a baseline period of six inspired gas conditions (9% O2, 12% O2, 21% O2, 100% O2, 5% CO2, and 10% CO2) followed by a superimposed period of forepaw stimulation. Significant stimulus-evoked fMRI responses were found in the primary somatosensory cortices. Relative fMRI responses to forepaw stimulation varied across gas conditions and were dependent on baseline physiology, whereas absolute fMRI responses were similar across moderate gas conditions (12% O2, 21% O2 100% O2, and 5% CO2) and were relatively independent of baseline physiology. Consistent with data obtained using well-established techniques, baseline and stimulus-evoked CMRO2 were invariant across moderate physiological perturbations thereby supporting a CMRO2-fMRI technique for non-invasive CMRO2 measurement. However, under 9% O2 and 10% CO2, stimulus-evoked CBF and BOLD were substantially reduced and the CMRO2 formalism appeared invalid, likely due to attenuated neurovascular coupling and/or a failure of the model under extreme physiological perturbations. These findings demonstrate that absolute fMRI measurements help distinguish neural from non-neural contributions to the fMRI signals and may lend a more accurate measure of brain activity during states of altered basal physiology. Moreover, since numerous pharmacologic agents, pathophysiological states, and psychiatric conditions alter baseline physiology independent of neural activity, these results have implications for neuroimaging studies using relative fMRI changes to map brain activity

Spectacular shrinking deficit: insights from multimodal magnetic resonance imaging after embolic middle cerebral artery occlusion in Sprague-Dawley rats

Almost no data is available on the serial changes in the brain after spectacular shrinking deficit (SSD) that may help understand this relatively rare clinical phenomenon. Quantitative diffusion-(DWI), perfusion-(PWI), T(1)-(T1WI), T(2)-weighted (T2WI), and functional magnetic resonance imaging (fMRI) were performed before, during, and up to 7 days after embolic middle cerebral artery occlusion (eMCAO) in male Sprague-Dawley rats (n=9). Region of interest (ROI) analysis was used to evaluate structural and functional MR signal changes within three ROIs defined by the apparent diffusion coefficient (ADC), cerebral blood flow (CBF) signatures, and final tissue viability. DWI, PWI, and T2WI lesion volumes were calculated using previously established viability thresholds and final infarct volumes ascertained with 2,3,5-triphenyltetrazolium chloride (TTC) staining. Serial MRI demonstrated spontaneous reperfusion of initially hypoperfused MCA regions accompanied by substantial reduction of initial ADC and CBF lesions and gradual recovery of neurological outcome. Recovery rates of CBF/ADC abnormalities differed among ROIs. Functional magnetic resonance imaging showed persistent tissue dysfunction after the recovery of the CBF/ADC lesions. This study may facilitate our understanding of the pathophysiological mechanisms by which early, spontaneous reperfusion affects tissue fate and neurological function

Imaging oxygen consumption in forepaw somatosensory stimulation in rats under isoflurane anesthesia

The cerebral metabolic rate of oxygen (CMRO2) was dynamically evaluated on a pixel-by-pixel basis in isoflurane-anesthetized and spontaneously breathing rats following graded electrical somatosensory forepaw stimulations (4, 6, and 8 mA). In contrast to alpha-chloralose, which is the most widely used anesthetic in forepaw-stimulation fMRI studies of rats under mechanical ventilation, isoflurane (1.1-1.2%) provided a stable anesthesia level over a prolonged period, without the need to adjust the ventilation volume/rate or sample blood gases. Combined cerebral blood flow signals (CBF) and blood oxygenation level-dependent (BOLD) fMRI signals were simultaneously measured with the use of a multislice continuous arterial spin labeling (CASL) technique (two-coil setup). CMRO2 was calculated using the biophysical BOLD model of Ogawa et al. (Proc Natl Acad Sci USA 1992;89:5951-5955). The stimulus-evoked BOLD percent changes at 4, 6, and 8 A were, respectively, 0.5% +/- 0.2%, 1.4% +/- 0.3%, and 2.0% +/- 0.3% (mean +/- SD, N = 6). The CBF percent changes were 23% +/- 6%, 58% +/- 9%, and 87% +/- 14%. The CMRO2 percent changes were 14% +/- 4%, 24% +/- 6%, and 43% +/- 11%. BOLD, CBF, and CMRO2 activations were localized to the forepaw somatosensory cortices without evidence of plateau for oxygen consumption, indicative of partial coupling of CBF and CMRO2. This study describes a useful forepaw-stimulation model for fMRI, and demonstrate that CMRO2 changes can be dynamically imaged on a pixel-by-pixel basis in a single setting with high spatiotemporal resolution

Normobaric hyperoxia delays perfusion/diffusion mismatch evolution, reduces infarct volume, and differentially affects neuronal cell death pathways after suture middle cerebral artery occlusion in rats

Normobaric hyperoxia (NBO) has been shown to extend the reperfusion window after focal cerebral ischemia. Employing diffusion (DWI)- and perfusion (PWI)-weighted magnetic resonance imaging (MRI), the effect of NBO (100% started at 30 mins after middle cerebral artery occlusion (MCAO)) on the spatiotemporal evolution of ischemia during and after permanent (pMCAO) and transient suture middle cerebral artery occlusion (tMCAO) was investigated (experiment 3). In two additional experiments, time window (experiment 1) and cell death pathways (experiment 2) were investigated in the pMCAO model. In experiment 1, NBO treatment reduced infarct volume at 24 h after pMCAO by 10% when administered for 3 h (P\u3e0.05) and by 44% when administered for 6 h (P\u3c0.05). In experiment 2, NBO acutely (390 mins, P\u3c0.05) reduced in situ end labeling (ISEL) positivity in the ipsilesional penumbra but increased contralesional necrotic as well as caspase-3-mediated apoptotic cell death. In experiment 3, CBF characteristics and CBF-derived lesion volumes did not differ between treated and untreated animals, whereas the apparent diffusion coefficient (ADC)-derived lesion volume essentially stopped progressing during NBO treatment, resulting in a persistent PWI/DWI mismatch that could be salvaged by delayed (3 h) reperfusion. In conclusion, NBO (1) acutely preserved the perfusion/diffusion mismatch without altering CBF, (2) significantly extended the time window for reperfusion, (3) induced lasting neuroprotection in permanent ischemia, and (4) although capable of reducing cell death in hypoperfused tissue it also induced cell death in otherwise unaffected areas. Our data suggest that NBO may represent a promising strategy for acute stroke treatment