Detection of Hyperexcitability by Functional Magnetic Resonance Imaging after Experimental Traumatic Brain Injury.

Diagnosis of ongoing epileptogenesis and associated hyperexcitability after brain injury is a major challenge. Given that increased neuronal activity in the brain triggers a blood oxygenation level-dependent (BOLD) response in functional magnetic resonance imaging (fMRI), we hypothesized that fMRI could be used to identify the brain area(s) with hyperexcitability during post-injury epileptogenesis. We applied fMRI to detect onset and spread of BOLD activation after pentylenetetrazol (PTZ)-induced seizures (PTZ, 30 mg/kg, intraperitoneally) in 16 adult male rats at 2 months after lateral fluid percussion (FPI)-induced traumatic brain injury (TBI). In sham-operated controls, onset of the PTZ-induced BOLD response was bilateral and first appeared in the cortex. After TBI, 5 of 9 (56%) rats exhibited ipsilateral perilesional cortical BOLD activation, followed by activation of the contralateral cortex. In 4 of 9 (44%) rats, onset of BOLD response was bilateral. Interestingly, latency from the PTZ injection to onset of the BOLD response increased in the following order: sham-operated controls (ipsilateral 132 ± 57 sec, contralateral 132 ± 57 sec; p > 0.05) < TBI with bilateral BOLD onset (ipsilateral 176 ± 54 sec, contralateral 178 ± 52 sec; p > 0.05) < TBI with ipsilateral BOLD onset (ipsilateral 406 ± 178 sec, contralateral 509 ± 140 sec; p < 0.05). Cortical lesion area did not differ between rats with ipsilateral versus bilateral BOLD onset (p > 0.05). In the group of rats with ipsilateral onset of PTZ-induced BOLD activation, none of the rats showed a robust bilateral thalamic BOLD response, only 1 of 5 rats had robust ipsilateral thalamic calcifications, and 4 of 5 rats had perilesional astrocytosis. These findings suggest the evolution of the epileptogenic zone in the perilesional cortex after TBI, which is sensitive to PTZ-induced hyperexcitability. Further studies are warranted to explore the evolution of thalamo-cortical pathology as a driver of epileptogenesis after lateral FPI.

Resting-state functional MRI as a tool for evaluating brain hemodynamic responsiveness to external stimuli in rats.

PURPOSE: Anesthesia is a major confounding factor in functional MRI (fMRI) experiments attributed to its effects on brain function. Recent evidence suggests that parameters obtained with resting-state fMRI (rs-fMRI) are coupled with anesthetic depth. Therefore, we investigated whether parameters obtained with rs-fMRI, such as functional connectivity (FC), are also directly related to blood-oxygen-level-dependent (BOLD) responses. METHODS: A simple rs-fMRI protocol was implemented in a pharmacological fMRI study to evaluate the coupling between hemodynamic responses and FC under five anesthetics (α-chloralose, isoflurane, medetomidine, thiobutabarbital, and urethane). Temporal change in the FC was evaluated at 1-hour interval. Supplementary forepaw stimulation experiments were also conducted. RESULTS: Under thiobutabarbital anesthesia, FC was clearly coupled with nicotine-induced BOLD responses. Good correlation values were also obtained under isoflurane and medetomidine anesthesia. The observations in the thiobutabarbital group were supported by forepaw stimulation experiments. Additionally, the rs-fMRI protocol revealed significant temporal changes in the FC in the α-chloralose, thiobutabarbital, and urethane groups. CONCLUSION: Our results suggest that FC can be used to estimate brain hemodynamic responsiveness to stimuli and evaluate the level and temporal changes of anesthesia. Therefore, analysis of the fMRI baseline signal may be highly valuable tool for controlling the outcome of preclinical fMRI experiments. Magn Reson Med 78:1136-1146, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Comparison of seven different anesthesia protocols for nicotine pharmacologic magnetic resonance imaging in rat.

Pharmacologic MRI (phMRI) is a non-invasive in vivo imaging method, which can evaluate the drug effects on the brain and provide complementary information to ex vivo techniques. The preclinical phMRI studies usually require anesthesia to reduce the motion and stress of the animals. The anesthesia, however, is a crucial part of the experimental design, as it may modulate the neural drug-induced (de)activation and hemodynamic coupling. Therefore, the aim of the present study was to address this methodologic question by performing phMRI experiments with five anesthetics (α-chloralose, isoflurane, medetomidine, thiobutabarbital, and urethane) and seven anesthesia protocols. Nicotine, a widely studied psychostimulant, was administered to rats while measuring blood oxygenation level-dependent (BOLD) signals. Notably different responses were observed depending on the anesthetic used. The highest responses were measured in urethane-anesthetized rats whereas the responses were hardly noticeable in α-chloralose group. As urethane is not commonly used in phMRI, hemodynamic coupling under urethane anesthesia was investigated with functional cerebral blood flow (CBF) and volume-weighted (CBVw) imaging, and simultaneous electrophysiologic and BOLD measurements. The BOLD, CBF, and CBVw measurements in response to nicotine were highly correlated (R(2) ≥ 0.70, p<0.001). BOLD values correlated well (R(2)=0.43, p<10(-6)) with local field potential (LFP) spectral power (13-70Hz) during pharmacologic stimulation. These findings indicate that urethane anesthesia combined with BOLD contrast provides a robust protocol for nicotine phMRI studies. As urethane has mild effects to individual receptor systems, and coupling between electrophysiologic activity and hemodynamic response is maintained, this anesthetic may also be suitable for other phMRI studies.

Posttraumatic epilepsy - disease or comorbidity?

Traumatic brain injury (TBI) can cause a myriad of sequelae depending on its type, severity, and location of injured structures. These can include mood disorders, posttraumatic stress disorder and other anxiety disorders, personality disorders, aggressive disorders, cognitive changes, chronic pain, sleep problems, motor or sensory impairments, endocrine dysfunction, gastrointestinal disturbances, increased risk of infections, pulmonary disturbances, parkinsonism, posttraumatic epilepsy, or their combinations. The progression of individual pathologies leading to a given phenotype is variable, and some progress for months. Consequently, the different post-TBI phenotypes appear within different time windows. In parallel with morbidogenesis, spontaneous recovery occurs both in experimental models and in human TBI. A great challenge remains; how can we dissect the specific mechanisms that lead to the different endophenotypes, such as posttraumatic epileptogenesis, in order to identify treatment approaches that would not compromise recovery?

Human bone marrow mesenchymal stem/stromal cells produce efficient localization in the brain and enhanced angiogenesis after intra-arterial delivery in rats with cerebral ischemia, but this is not translated to behavioral recovery.

Intravascular cell therapy is a promising approach for the treatment of stroke. However, high accumulation of cells to lungs and other filtering organs is a major concern after intravenous (i.v.) cell transplantation. This can be circumvented by intra-arterial (i.a.) cell infusion, which improves homing of cells to the injured brain. We studied the effect of i.a. delivery of human bone marrow-derived mesenchymal cells (BMMSCs) on behavioral and histological outcome in rats after middle cerebral artery occlusion (MCAO). Sixty male Wistar rats were subjected to transient MCAO (60 min) or sham-operation. BMMSCs (1×10(6)) were infused into the external carotid artery on postoperative day 2 or 7. Histology performed after a 42-day follow-up did not detect any human cells (MAB1281) in the ischemic brain. Endothelial cell staining with RECA-1 revealed a significant increase in the number of blood vessels in the perilesional cortex in MCAO rats treated with cells on postoperative day 7. Behavioral recovery as assessed in three tests, sticky label, cylinder and Montoya's staircase, was not improved by human BMMSCs during the follow-up. In conclusion, human BMMSCs did not improve functional recovery in MCAO rats despite effective initial homing to the ischemic hemisphere and enhanced angiogenesis, when strict behavioral tests not affected by repeated testing and compensation were utilized.

Gait impairment in a rat model of focal cerebral ischemia.

The availability of proper tests for gait evaluation following cerebral ischemia in rats has been limited. The automated, quantitative CatWalk system, which was initially designed to measure gait in models of spinal cord injury, neuropathic pain, and peripheral nerve injury, is said to be a useful tool for the study of motor impairment in stroke animals. Here we report our experiences of using CatWalk XT with rats subjected to transient middle cerebral artery occlusion (MCAO), during their six-week followup. Large corticostriatal infarct was confirmed by MRI in all MCAO rats, which was associated with severe sensorimotor impairment. In contrast, the gait impairment was at most mild, which is consistent with seemingly normal locomotion of MCAO rats. Many of the gait parameters were affected by body weight, walking speed, and motivation despite the use of a goal box. In addition, MCAO rats showed bilateral compensation, which was developed to stabilize proper locomotion. All of these interferences may confound the data interpretation. Taken together, the translational applicability of CatWalk XT in evaluating motor impairment and treatment efficacy remains to be limited at least in rats with severe corticostriatal infarct and loss of body weight.

Monitoring functional impairment and recovery after traumatic brain injury in rats by FMRI.

The present study was designed to test a hypothesis that functional magnetic resonance imaging (fMRI) can be used to monitor functional impairment and recovery after moderate experimental traumatic brain injury (TBI). Moderate TBI was induced by lateral fluid percussion injury in adult rats. The severity of brain damage and functional recovery in the primary somatosensory cortex (S1) was monitored for up to 56 days using fMRI, cerebral blood flow (CBF) by arterial spin labeling, local field potential measurements (LFP), behavioral assessment, and histology. All the rats had reduced blood-oxygen-level-dependent (BOLD) responses during the 1st week after trauma in the ipsilateral S1. Forty percent of these animals showed recovery of the BOLD response during the 56 day follow-up. Unexpectedly, no association was found between the recovery in BOLD response and the volume of the cortical lesion or thalamic neurodegeneration. Instead, the functional recovery occurred in rats with preserved myelinated fibers in layer VI of S1. This is, to our knowledge, the first study demonstrating that fMRI can be used to monitor post-TBI functional impairment and consequent spontaneous recovery. Moreover, the BOLD response was associated with the density of myelinated fibers in the S1, rather than with neurodegeneration. The present findings encourage exploration of the usefulness of fMRI as a noninvasive prognostic biomarker for human post-TBI outcomes and therapy responses.

Simultaneous BOLD fMRI and local field potential measurements during kainic acid-induced seizures.

PURPOSE: To investigate how kainic acid-induced epileptiform activity is related to hemodynamic changes probed by blood oxygenation level-dependent functional magnetic resonance imaging (BOLD fMRI). METHODS: Epileptiform activity was induced with kainic acid (KA) (10 mg/kg, i.p.), and simultaneous fMRI at 7 Tesla, and deep electrode local field potential (LFP) recordings were performed from the right hippocampus in awake and medetomidine-sedated adult Wistar rats. KEY FINDINGS: Recurrent seizure activity induced by KA was detected in LFP both in medetomidine-sedated and awake rats, even though medetomidine sedation reduced the mean duration of individual seizures as compared to awake rats (33 ± 24 and 46 ± 34 s, respectively, mean ± SD p < 0.01). KA administration also triggered robust positive BOLD responses bilaterally in the hippocampus both in awake and medetomidine-sedated rats; however, in both animal groups some of the seizures detected in LFP recording did not cause detectable BOLD signal change. SIGNIFICANCE: Our data suggest that medetomidine sedation can be used for simultaneous fMRI and electrophysiologic studies of normal and epileptic brain function, even though seizure duration after medetomidine administration was shorter than that in awake animals. The results also indicate that neuronal activity and BOLD response can become decoupled during recurrent kainic acid-induced seizures, which may have implications to interpretation of fMRI data obtained during prolonged epileptiform activity.

Evoked local field potentials can explain temporal variation in blood oxygenation level-dependent responses in rat somatosensory cortex.

The aim of this study was to explain the temporal variations between subjects in the blood oxygenation level-dependent (BOLD) response. Somatosensory responses were elicited with the electrical forepaw stimulus at a frequency of 10 Hz in urethane-anesthetized rats, and functional magnetic resonance imaging (fMRI) with BOLD contrast and local field potential (LFP) measurements were performed simultaneously. BOLD fMRI activation was evaluated by two different models, one based on the stimulus paradigm (the block model) and the other on the simultaneously measured evoked LFP responses. In the initial analysis, the LFP model captured the BOLD activation in the primary somatosensory cortex in all cases, and the block model in 10 of 12 rats. A statistical comparison of the two models revealed that the LFP-derived model was able to explain additional BOLD variation over the block model in the somatosensory cortex in nine of 12 rats. These results suggest that there is more information regarding neuronal activity in the BOLD signal than can be exploited using the block model alone. Furthermore, the hemodynamic coupling remains unchanged in the case of temporally variable BOLD signals.

Simultaneous fMRI and local field potential measurements during epileptic seizures in medetomidine-sedated rats using raser pulse sequence.

Simultaneous electrophysiological and functional magnetic resonance imaging measurements of animal models of epilepsy are methodologically challenging, but essential to better understand abnormal brain activity and hemodynamics during seizures. In this study, functional magnetic resonance imaging of medetomidine-sedated rats was performed using novel rapid acquisition by sequential excitation and refocusing (RASER) fast imaging pulse sequence and simultaneous local field potential measurements during kainic acid-induced seizures. The image distortion caused by the hippocampal-measuring electrode was clearly seen in echo planar imaging images, whereas no artifact was seen in RASER images. Robust blood oxygenation level-dependent responses were observed in the hippocampus during kainic acid-induced seizures. The recurrent epileptic seizures were detected in the local field potential signal after kainic acid injection. The presented combination of deep electrode local field potential measurements and functional magnetic resonance imaging under medetomidine anesthesia, which does not significantly suppress kainic acid-induced seizures, provides a unique tool for studying abnormal brain activity in rats.

Coupling between simultaneously recorded BOLD response and neuronal activity in the rat somatosensory cortex.

Understanding the link between the hemodynamic response and the underlying neuronal activity is important for interpreting functional magnetic resonance (fMRI) signals in human and animal studies. Simultaneous electrophysiological and functional imaging measurements provide a knowledge of information processing and communication in the brain with high spatial and temporal resolution. In this study, a range of neural and blood oxygenation level-dependent (BOLD) responses were elicited in the rat somatosensory cortex by changing the type of anesthesia (urethane or alpha-chloralose) and the electrical forepaw stimulus frequency (1-15 Hz). Duration of the stimulus was 30 s. Electrical local field potential and BOLD fMRI responses were recorded simultaneously. Under urethane anesthesia, integrated neural activity and BOLD responses increased with increasing stimulus frequency up to 11 Hz, after which both responses plateaued. In contrast, in alpha-chloralose-anesthetized rats both responses were measurable only at 1 and 3 Hz. Although neuronal and BOLD responses were nonlinear as a function of frequency over the 1 to 15 Hz stimulation range under both anesthetics, tight neural-hemodynamic coupling was observed independently of the anesthetic agent. Anesthetic agents influence neuronal activity in a different manner, but the relationship of neuronal activity and BOLD response remains the same.

Contribution of a single CA3 neuron to network synchrony.

Oscillations at theta (3-8 Hz) and gamma (30-80 Hz) frequencies co-occur during arousal, exploration, and rapid eye movement sleep and relate to information processing underlying learning and memory within neuronal networks. In hippocampus, gamma and theta frequency oscillations are associated with modification of synaptic weights, spatial learning, and short-term memory. These oscillations are referred to as network phenomena and, thereby, the role of single neuron oscillations in the generation of neuronal networks remains unclear. We report that an individual CA3 pyramidal cell can activate the CA1 neuronal network in vivo in rat hippocampus using electrical stimulations with simultaneous intracellular gamma and extracellular theta and slow (0.5-1 Hz) frequencies. These results suggest that an individual pyramidal cell can contribute to self-organization of a neuronal small-scale network.

Manganese-enhanced magnetic resonance imaging of mossy fiber plasticity in vivo.

Mn(2+)-enhanced magnetic resonance imaging (MEMRI) was used to characterize activity-dependent plasticity in the mossy fiber pathway after intraperitoneal kainic acid (KA) injection. Enhancement of the MEMRI signal in the dentate gyrus and the CA3 subregion of the hippocampus was evident 3 to 5 days after injection of MnCl(2) into the entorhinal cortex both in control and KA-injected rats. In volume-rendered three-dimensional reconstructions, Mn(2+)-induced signal enhancement revealed the extent of the mossy fiber pathway throughout the septotemporal axis of the dentate gyrus. An increase in the number of Mn(2+)-enhanced pixels in the dentate gyrus and CA3 subfield of rats with KA injection correlated (P < 0.05) with histologically verified mossy fiber sprouting. These data demonstrate that MEMRI can be used to detect specific changes at the cellular level during activity-dependent plasticity in vivo. The present findings also suggest that MEMRI signal changes can serve as an imaging marker of epileptogenesis.