Temporal trajectories of normal myelination and axonal development assessed by quantitative macromolecular and diffusion MRI: Ultrastructural and immunochemical validation in a rabbit model.

INTRODUCTION: Quantitative and non-invasive measures of brain myelination and maturation during development are of great importance to both clinical and translational research communities. While the metrics derived from diffusion tensor imaging, are sensitive to developmental changes and some pathologies, they remain difficult to relate to the actual microstructure of the brain tissue. The advent of advanced model-based microstructural metrics requires histological validation. The purpose of the study was to validate novel, model-based MRI techniques, such as macromolecular proton fraction mapping (MPF) and neurite orientation and dispersion indexing (NODDI), against histologically derived indexes of myelination and microstructural maturation at various stages of development. METHODS: New Zealand White rabbit kits underwent serial in-vivo MRI examination at postnatal days 1, 5, 11, 18, and 25, and as adults. Multi-shell, diffusion-weighted experiments were processed to fit NODDI model to obtain estimates, intracellular volume fraction (ICVF) and orientation dispersion index (ODI). Macromolecular proton fraction (MPF) maps were obtained from three source (MT-, PD-, and T1-weighted) images. After MRI sessions, a subset of animals was euthanized and regional samples of gray and white matter were taken for western blot analysis, to determine myelin basic protein (MBP), and electron microscopy, to estimate axonal, myelin fractions and g-ratio. RESULTS: MPF of white matter regions showed a period of fast growth between P5 and P11 in the internal capsule, with a later onset in the corpus callosum. This MPF trajectory was in agreement with levels of myelination in the corresponding brain region, as assessed by western blot and electron microscopy. In the cortex, the greatest increase of MPF occurred between P18 and P26. In contrast, myelin, according to MBP western blot, saw the largest hike between P5 and P11 in the sensorimotor cortex and between P11 and P18 in the frontal cortex, which then seemingly plateaued after P11 and P18 respectively. G-ratio by MRI markers decreased with age in the white matter. However, electron microscopy suggest a relatively stable g-ratio throughout development. CONCLUSION: Developmental trajectories of MPF accurately reflected regional differences of myelination rate in different cortical regions and white matter tracts. MRI-derived estimation of g-ratio was inaccurate during early development, likely due to the overestimation of axonal volume fraction by NODDI due to the presence of a large proportion of unmyelinated axons.

Early Development of the GABAergic System and the Associated Risks of Neonatal Anesthesia.

Human and animal studies have elucidated the apparent neurodevelopmental effects resulting from neonatal anesthesia. Observations of learning and behavioral deficits in children, who were exposed to anesthesia early in development, have instigated a flurry of studies that have predominantly utilized animal models to further interrogate the mechanisms of neonatal anesthesia-induced neurotoxicity. Specifically, while neonatal anesthesia has demonstrated its propensity to affect multiple cell types in the brain, it has shown to have a particularly detrimental effect on the gamma aminobutyric acid (GABA)ergic system, which contributes to the observed learning and behavioral deficits. The damage to GABAergic neurons, resulting from neonatal anesthesia, seems to involve structure-specific changes in excitatory-inhibitory balance and neurovascular coupling, which manifest following a significant interval after neonatal anesthesia exposure. Thus, to better understand how neonatal anesthesia affects the GABAergic system, we first review the early development of the GABAergic system in various structures that have been the focus of neonatal anesthesia research. This is followed by an explanation that, due to the prolonged developmental curve of the GABAergic system, the entirety of the negative effects of neonatal anesthesia on learning and behavior in children are not immediately evident, but instead take a substantial amount of time (years) to fully develop. In order to address these concerns going forward, we subsequently offer a variety of in vivo methods which can be used to record these delayed effects.

Oscillations and concentration dynamics of brain tissue oxygen in neonates and adults.

The brain is a metabolically demanding organ and its health directly depends on brain oxygen dynamics to prevent hypoxia and ischemia. Localized brain tissue oxygen is characterized by a baseline level combined with spontaneous oscillations. These oscillations are attributed to spontaneous changes of vascular tone at the level of arterioles and their frequencies depend on age. Specifically, lower frequencies are more typical for neonates than for adults. We have built a mathematical model which analyses the diffusion abilities of oxygen based on the frequency of source brain oxygen oscillations and neuronal demand. We have found that a lower frequency of spontaneous oscillations of localized brain tissue oxygen can support higher amplitudes of oxygen concentration at areas distant from a source relative to oscillations at higher frequencies. Since hypoxia and ischemia are very common events during early development and the neurovascular unit is underdeveloped in neonates, our results indicate that lower frequency oxygen oscillations can represent an effective passive method of neonatal brain protection against hypoxia. These results can have a potential impact on future studies aiming to find new treatment strategies for brain ischemia.

Impact of anesthesia exposure in early development on learning and sensory functions.

Each year, millions of children undergo anesthesia, and both human and animal studies have indicated that exposure to anesthesia at an early age can lead to neuronal damage and learning deficiency. However, disorders of sensory functions were not reported in children or animals exposed to anesthesia during infancy, which is surprising, given the significant amount of damage to brain tissue reported in many animal studies. In this review, we discuss the relationship between the systems in the brain that mediate sensory input, spatial learning, and classical conditioning, and how these systems could be affected during anesthesia exposure. Based on previous reports, we conclude that anesthesia can induce structural, functional, and compensatory changes in both sensory and learning systems. Changes in myelination following anesthesia exposure were observed as well as the neurodegeneration in the gray matter across variety of brain regions. Disproportionate cell death between excitatory and inhibitory cells induced by anesthesia exposure can lead to a long-term shift in the excitatory/inhibitory balance, which affects both learning-specific networks and sensory systems. Anesthesia may directly affect synaptic plasticity which is especially critical to learning acquisition. However, sensory systems appear to have better ability to compensate for damage than learning-specific networks.

Role of the inhibitory system in shaping the BOLD fMRI response.

The shape of the blood oxygenation level dependent (BOLD) functional magnetic resonance imaging (fMRI) signal can vary considerably even across structures of the same sensory pathway. Here, we characterized the temporal behavior of the stimulus-evoked BOLD response in the primary cortical and subcortical regions of the visual and somatosensory whisker systems in awake rabbits. Despite similar BOLD responses in the thalamic nuclei, considerable differences in shape and duration emerged between the sensory cortices. Whereas the BOLD response in the whisker barrel cortex (WBC) was non-adaptive, BOLD in the visual cortex (V1) showed adaptation similar to simultaneously-recorded LFP and single unit activity. Analysis of baseline neuronal activity revealed significantly lower firing rates in V1 vs. WBC. We hypothesized that these changes point to region-dependent differences in the inhibitory systems which shape the hemodynamic response in each structure. To test the effect of neuronal baseline level inhibition on the BOLD signal shape, we locally injected the GABAA agonist muscimol in WBC. Adaptation emerged in the BOLD response after injection, along with an overall decrease in baseline firing rate. These findings point to the importance of region-specific inhibitory shaping in determining the temporal behavior of the BOLD response in different brain areas.

The effect of sevoflurane and isoflurane anesthesia on single unit and local field potentials.

Volatile general anesthetics are used commonly in adults and children, yet their mechanisms of action are complex and the changes in single unit firing and synaptic activity that underlie the broad decreases in neuronal activity induced by these drugs have not been well characterized. Capturing such changes throughout the anesthesia process is important for comparing the effects of different anesthetics and gaining a better understanding of their mechanisms of action and their impact on different brain regions. Using chronically implanted electrodes in the rabbit somatosensory cortex, we compared the effects of two common general anesthetics, isoflurane, and sevoflurane, on cortical neurons. Single unit activity and local field potentials (LFP) were recorded continuously before and during anesthetic delivery at 1 MAC, as well as during recovery. Our findings show that although isoflurane and sevoflurane belong to the same class of volatile general anesthetics, their effects upon cortical single units and LFP were quite different. Overall, the suppression of neuronal firing was greater and more uniform under sevoflurane. Moreover, the changes in LFP frequency bands suggest that effect of anesthesia upon beta oscillations does not necessarily depend on the level of single unit activity, but rather on the changes in GABA/glutamate neurotransmission induced by each drug.

Computed Tomography in the Diagnosis of Classical Trigeminal Neuralgia.

OBJECTIVE: This study aims to characterize the pathogenesis of neurovascular conflict in trigeminal neuralgia (TN) with the goal of producing a reliable diagnostic method. METHODS: Data were obtained during microvascular decompression surgery. The normal relationship between the superior cerebellar artery (SCA) and the trigeminal nerve root also was characterized in postmortem cases. Furthermore, SCA was imaged with high-resolution computed tomography angiography. RESULTS: Our results indicate that contact between SCA and the trigeminal nerve root is present in both TN and non-TN cases, but in patients with TN, the apex of SCA loop was always at the bottom of the upper edge of the trigeminal root forming the state of neurovascular conflict. High-resolution angiography supported the intraoperative results. CONCLUSIONS: These findings indicate a clear difference between neurovascular contact and conflict and point to the use of high-resolution computed tomography visualization of the position of SCA loop apex relative to the trigeminal nerve root as a primary diagnostic method.

Blood oxygenation level dependent signal and neuronal adaptation to optogenetic and sensory stimulation in somatosensory cortex in awake animals.

The adaptation of neuronal responses to stimulation, in which a peak transient response is followed by a sustained plateau, has been well-studied. The blood oxygenation level dependent (BOLD) functional magnetic resonance imaging (fMRI) signal has also been shown to exhibit adaptation on a longer time scale. However, some regions such as the visual and auditory cortices exhibit significant BOLD adaptation, whereas other such as the whisker barrel cortex may not adapt. In the sensory cortex a combination of thalamic inputs and intracortical activity drives hemodynamic changes, although the relative contributions of these components are not entirely understood. The aim of this study is to assess the role of thalamic inputs vs. intracortical processing in shaping BOLD adaptation during stimulation in the somatosensory cortex. Using simultaneous fMRI and electrophysiology in awake rabbits, we measured BOLD, local field potentials (LFPs), single- and multi-unit activity in the cortex during whisker and optogenetic stimulation. This design allowed us to compare BOLD and haemodynamic responses during activation of the normal thalamocortical sensory pathway (i.e., both inputs and intracortical activity) vs. the direct optical activation of intracortical circuitry alone. Our findings show that whereas LFP and multi-unit (MUA) responses adapted, neither optogenetic nor sensory stimulation produced significant BOLD adaptation. We observed for both paradigms a variety of excitatory and inhibitory single unit responses. We conclude that sensory feed-forward thalamic inputs are not primarily responsible for shaping BOLD adaptation to stimuli; but the single-unit results point to a role in this behaviour for specific excitatory and inhibitory neuronal sub-populations, which may not correlate with aggregate neuronal activity.

Spontaneous Fluctuations of PO2 in the Rabbit Somatosensory Cortex.

In many tissues, PO2 fluctuates spontaneously with amplitudes of a few mmHg. Here we further characterized these oscillations. PO2 recordings were made from the whisker barrel cortex of six rabbits with acutely or chronically placed polarographic electrodes. Measurements were made while rabbits were awake and while anesthetized with isoflurane, during air breathing, and during 100% oxygen inspiration. In awake rabbits, 90% of the power was between 0 and 20 cycles per minute (cpm), not uniformly distributed over this range, but with a peak frequently near 10 cpm. This was much slower than heart or respiratory rhythms and is similar to the frequency content observed in other tissues. During hyperoxia, total power was higher than during air-breathing, and the dominant frequencies tended to shift toward lower values (0-10 cpm). These observations suggest that at least the lower frequency fluctuations represent efforts by the circulation to regulate local PO2. There were no consistent changes in total power during 0.5 or 1.5% isoflurane anesthesia, but the power shifted to lower frequencies. Thus, both hyperoxia and anesthesia cause characteristic, but distinct, changes in spontaneous fluctuations. These PO2 fluctuations may be caused by vasomotion, but other factors cannot be ruled out.

Volume effect of localized injection in functional MRI and electrophysiology.

PURPOSE: The local injection of neurotransmitter agonists and antagonists to modulate recorded neurons in awake animals has long been an important and widely used technique in neuroscience. Combined with functional magnetic resonance imaging (fMRI) and simultaneous electrophysiology, local injection enables the study of specific brain regions under precise modulations of their neuronal activity. However, localized injections are often accompanied by mechanical displacement of the tissue, known as volume effect (VE), which can induce changes in electrophysiological recordings as well as artifacts that are particular to fMRI studies. METHODS: We characterize the changes produced by VE in an agarose phantom as well as during stimulus-evoked and resting-state fMRI and simultaneously acquired electrophysiology in awake rabbits. RESULTS: Our results demonstrate that localized injection can produce significant intensity changes in fMRI data, even while effects on electrophysiological recordings are minimized. These changes are localized to the vicinity of the injection needle and diminish over time due to diffusion of the injected volume. CONCLUSION: Sufficient time should be allowed for drug diffusion to ensure stable results, particularly for resting-state fMRI experiments.

Functional Deficiency of Interneurons and Negative BOLD fMRI Response.

The functional deficiency of the inhibitory system typically appears during development and can progress to psychiatric disorders or epilepsy, depending on its severity, in later years. It is known that interneurons, the major source of GABAergic inhibition in the cerebral cortex, can make direct connections with arterioles and participate in the regulation of vasomotion. The goal of this study was to mimic the functional deficiency of interneurons through the use of localized microinjections of the GABA antagonist, picrotoxin, in such a concentration that it did not elicit epileptiform neuronal activity. First, we recorded the dynamics of resting-state neuronal activity in response to picrotoxin injections in the somatosensory cortex of an awake rabbit; second, we assessed the altered neuronal and hemodynamic responses to whisker stimulation using BOLD fMRI and electrophysiology recordings; third, we evaluated brain tissue oxygen levels before and after picrotoxin injection. Our results showed that neuronal activity typically increased after picrotoxin administration, the BOLD responses to stimulation became negative, and the oxygen response was nearly abolished. Vasoconstriction during the resting baseline was not observed. These results indicate that picrotoxin provoked imbalanced hemodynamics either due to increased neuronal activity, decreased vascular response, or a combination of both.

Electric Field Effects on Brain Activity: Implications for Epilepsy and Burst Suppression.

Electric fields are now considered a major mechanism of epileptiform activity. However, it is not clear if another electrophysiological phenomenon, burst suppression, utilizes the same mechanism for its bursting phase. Thus, the purpose of this study was to compare the role of ephaptic coupling-the recruitment of neighboring cells via electric fields-in generating bursts in epilepsy and burst suppression. We used local injections of the GABA-antagonist picrotoxin to elicit epileptic activity and a general anesthetic, sevoflurane, to elicit burst suppression in rabbits. Then, we applied an established computational model of pyramidal cells to simulate neuronal activity in a 3-dimensional grid, with an additional parameter to trigger a suppression phase based on extra-cellular calcium dynamics. We discovered that coupling via electric fields was sufficient to produce bursting in scenarios where inhibitory control of excitatory neurons was sufficiently low. Under anesthesia conditions, bursting occurs with lower neuronal recruitment in comparison to seizures. Our model predicts that due to the effect of electric fields, the magnitude of bursts during seizures should be roughly 2-3 times the magnitude of bursts that occur during burst suppression, which is consistent with our in vivo experimental results. The resulting difference in magnitude between bursts during anesthesia and epileptiform bursts reflects the strength of the electric field effect, which suggests that burst suppression and epilepsy share the same ephaptic coupling mechanism.

Neonatal Anesthesia and Oxidative Stress.

Neonatal anesthesia, while often essential for surgeries or imaging procedures, is accompanied by significant risks to redox balance in the brain due to the relatively weak antioxidant system in children. Oxidative stress is characterized by concentrations of reactive oxygen species (ROS) that are elevated beyond what can be accommodated by the antioxidant defense system. In neonatal anesthesia, this has been proposed to be a contributing factor to some of the negative consequences (e.g., learning deficits and behavioral abnormalities) that are associated with early anesthetic exposure. In order to assess the relationship between neonatal anesthesia and oxidative stress, we first review the mechanisms of action of common anesthetic agents, the key pathways that produce the majority of ROS, and the main antioxidants. We then explore the possible immediate, short-term, and long-term pathways of neonatal-anesthesia-induced oxidative stress. We review a large body of literature describing oxidative stress to be evident during and immediately following neonatal anesthesia. Moreover, our review suggests that the short-term pathway has a temporally limited effect on oxidative stress, while the long-term pathway can manifest years later due to the altered development of neurons and neurovascular interactions.

Function and development of interneurons involved in brain tissue oxygen regulation.

The regulation of oxygen in brain tissue is one of the most important fundamental questions in neuroscience and medicine. The brain is a metabolically demanding organ, and its health directly depends on maintaining oxygen concentrations within a relatively narrow range that is both sufficiently high to prevent hypoxia, and low enough to restrict the overproduction of oxygen species. Neurovascular interactions, which are responsible for oxygen delivery, consist of neuronal and glial components. GABAergic interneurons play a particularly important role in neurovascular interactions. The involvement of interneurons extends beyond the perspective of inhibition, which prevents excessive neuronal activity and oxygen consumption, and includes direct modulation of the microvasculature depending upon their sub-type. Namely, nitric oxide synthase-expressing (NOS), vasoactive intestinal peptide-expressing (VIP), and somatostatin-expressing (SST) interneurons have shown modulatory effects on microvessels. VIP interneurons are known to elicit vasodilation, SST interneurons typically cause vasoconstriction, and NOS interneurons have to propensity to induce both effects. Given the importance and heterogeneity of interneurons in regulating local brain tissue oxygen concentrations, we review their differing functions and developmental trajectories. Importantly, VIP and SST interneurons display key developmental milestones in adolescence, while NOS interneurons mature much earlier. The implications of these findings point to different periods of critical development of the interneuron-mediated oxygen regulatory systems. Such that interference with normal maturation processes early in development may effect NOS interneuron neurovascular interactions to a greater degree, while insults later in development may be more targeted toward VIP- and SST-mediated mechanisms of oxygen regulation.

Brain tissue oxygen dynamics while mimicking the functional deficiency of interneurons.

The dynamic interaction between excitatory and inhibitory activity in the brain is known as excitatory-inhibitory balance (EIB). A significant shift in EIB toward excitation has been observed in numerous pathological states and diseases, such as autism or epilepsy, where interneurons may be dysfunctional. The consequences of this on neurovascular interactions remains to be elucidated. Specifically, it is not known if there is an elevated metabolic consumption of oxygen due to increased excitatory activity. To investigate this, we administered microinjections of picrotoxin, a gamma aminobutyric acid (GABA) antagonist, to the rabbit cortex in the awake state to mimic the functional deficiency of GABAergic interneurons. This caused an observable shift in EIB toward excitation without the induction of seizures. We used chronically implanted electrodes to measure both neuronal activity and brain tissue oxygen concentrations (PO2) simultaneously and in the same location. Using a high-frequency recording rate for PO2, we were able to detect two important phenomena, (1) the shift in EIB led to a change in the power spectra of PO2 fluctuations, such that higher frequencies (8-15 cycles per minute) were suppressed and (2) there were brief periods (dips with a duration of less than 100 ms associated with neuronal bursts) when PO2 dropped below 10 mmHg, which we defined as the threshold for hypoxia. The dips were followed by an overshoot, which indicates either a rapid vascular response or decrease in oxygen consumption. Our results point to the essential role of interneurons in brain tissue oxygen regulation in the resting state.

Occlusion of activity dependent synaptic plasticity by late hypoxic long term potentiation after neonatal intermittent hypoxia.

To elucidate the mechanisms of memory impairment after chronic neonatal intermittent hypoxia (IH), we employed a mice model of severe IH administered at postnatal days 3 to 7. Since prior studies in this model did not demonstrate increased cell death, our primary hypothesis was that IH causes a functional disruption of synaptic plasticity in hippocampal neurons. In vivo recordings of Schaffer collateral stimulation-induced synaptic responses during and after IH in the CA1 region of the hippocampus revealed pathological late phase hypoxic long term potentiation (hLTP) (154%) that lasted more than four hours and could be reversed by depotentiation with low frequency stimulation (LFS), or abolished by NMDA and PKA inhibitors (MK-801 and CMIQ). Furthermore, late phase hLTP partially occluded normal physiological LTP (pLTP) four hours after IH. Early and late hLTP phases were induced by neuronal depolarization and Ca2+ influx, determined with manganese enhanced fMRI, and had increased both AMPA and NMDA - mediated currents. This was consistent with mechanisms of pLTP in neonates and also consistent with mechanisms of ischemic LTP described in vitro with OGD in adults. A decrease of pLTP was also recorded on hippocampal slices obtained 2 days after IH. This decrease was ameliorated by MK-801 injections prior to each IH session and restored by LFS depotentiation. Occlusion of pLTP and the observed decreased proportion of NMDA-only silent synapses after neonatal hLTP may explain long term memory, behavioral deficits and abnormal synaptogenesis and pruning following neonatal IH.

Behavior and Regional Cortical BOLD Signal Fluctuations Are Altered in Adult Rabbits After Neonatal Volatile Anesthetic Exposure.

Neonatal and infant exposure to volatile anesthetics has been associated with long-term learning, memory, and behavioral deficits. Although early anesthesia exposure has been linked to a number of underlying structural abnormalities, functional changes associated with these impairments remain poorly understood. To investigate the relationship between functional alteration in neuronal circuits and learning deficiency, resting state functional MRI (rsfMRI) connectivity was examined in adolescent rabbits exposed to general anesthesia as neonates (1 MAC isoflurane for 2 h on postnatal days P8, P11, and P14) and unanesthetized controls before and after training with a trace eyeblink classical conditioning (ECC) paradigm. Long-range connectivity was measured between several key regions of interest (ROIs), including primary and secondary somatosensory cortices, thalamus, hippocampus, and cingulate. In addition, metrics of regional BOLD fluctuation amplitudes and coherence, amplitude of low-frequency fluctuation (ALFF), fractional ALFF (fALFF), and regional homogeneity (ReHo) were calculated. Our results showed that the trace ECC learning rate was significantly lower in the anesthesia-exposed group. No anesthesia-related changes in long-range connectivity, fALFF, or ReHo were found between any ROIs. However, ALFF was significantly higher in anesthesia-exposed rabbits in the primary and secondary somatosensory cortices, and ALFF in those areas was a significant predictor of the learning performance for trace ECC. The absence of anesthesia-related changes in long-range thalamocortical connectivity indicates that functional thalamocortical input is not affected. Higher ALFF in the somatosensory cortex may indicate the developmental disruption of cortical neuronal circuits after neonatal anesthesia exposure, including excessive neuronal synchronization that may underlie the observed cognitive deficits.

Effects of neonatal isoflurane anesthesia exposure on learning-specific and sensory systems in adults.

Millions of children undergo general anesthesia each year, and animal and human studies have indicated that exposure to anesthesia at an early age can impact neuronal development, leading to behavioral and learning impairments that manifest later in childhood and adolescence. Here, we examined the effects of isoflurane, a commonly-used general anesthetic, which was delivered to newborn rabbits. Trace eyeblink classical conditioning was used to assess the impact of neonatal anesthesia exposure on behavioral learning in adolescent subjects, and a variety of MRI techniques including fMRI, MR volumetry, spectroscopy and DTI captured functional, metabolic, and structural changes in key regions of the learning and sensory systems associated with anesthesia-induced learning impairment. Our results demonstrated a wide array of changes that were specific to anesthesia-exposed subjects, which supports previous studies that have pointed to a link between early anesthesia exposure and the development of learning and behavioral deficiencies. These findings point to the need for caution in avoiding excessive use of general anesthesia in young children and neonates.