Cognitive Assessment and Rehabilitation of subjects with Traumatic Brain Injury

This thesis regards the study and the development of new cognitive assessment and rehabilitation techniques of subjects with traumatic brain injury (TBI). In particular, this thesis i) provides an overview about the state of art of this new assessment and rehabilitation technologies, ii) suggests new methods for the assessment and rehabilitation and iii) contributes to the explanation of the neurophysiological mechanism that is involved in a rehabilitation treatment. Some chapters provide useful information to contextualize TBI and its outcome; they describe the methods used for its assessment/rehabilitation. The other chapters illustrate a series of experimental studies conducted in healthy subjects and TBI patients that suggest new approaches to assessment and rehabilitation. The new proposed approaches have in common the use of electroencefalografy (EEG). EEG was used in all the experimental studies with a different purpose, such as diagnostic tool, signal to command a BCI-system, outcome measure to evaluate the effects of a treatment, etc. The main achieved results are about: i) the study and the development of a system for the communication with patients with disorders of consciousness. It was possible to identify a paradigm of reliable activation during two imagery task using EEG signal or EEG and NIRS signal; ii) the study of the effects of a neuromodulation technique (tDCS) on EEG pattern. This topic is of great importance and interest. The emerged founding showed that the tDCS can manipulate the cortical network activity and through the research of optimal stimulation parameters, it is possible move the working point of a neural network and bring it in a condition of maximum learning. In this way could be possible improved the performance of a BCI system or to improve the efficacy of a rehabilitation treatment, like neurofeedback.

Chronic excessive erythrocytosis induces endothelial activation and damage in mouse brain

Excessive erythrocytosis results in severely increased blood viscosity, which may have significant detrimental effects on endothelial cells and, ultimately, function of the vascular endothelium. Because blood-brain barrier stability is crucial for normal physiological function, we used our previously characterized erythropoietin-overexpressing transgenic (tg6) mouse line (which has a hematocrit of 0.8-0.9) to investigate the effect of excessive erythrocytosis on vessel number, structure, and integrity in vivo. These mice have abnormally high levels of nitric oxide (NO), a potent proinflammatory molecule, suggesting altered vascular permeability and function. In this study, we observed that brain vessel density of tg6 mice was significantly reduced (16%) and vessel diameter was significantly increased (15%) compared with wild-type mice. Although no significant increases in vascular permeability under normoxic or acute hypoxic conditions (8% O2 for 4 h) were detected, electron-microscopic analysis revealed altered morphological characteristics of the tg6 endothelium. Tg6 brain vascular endothelial cells appeared to be activated, with increased luminal protrusions reminiscent of ongoing inflammatory processes. Consistent with this observation, we detected increased levels of intercellular adhesion molecule-1 and von Willebrand factor, markers of endothelial activation and damage, in brain tissue. We propose that chronic excessive erythrocytosis and sustained high hematocrit cause endothelial damage, which may, ultimately, increase susceptibility to vascular disease.

Determination of histopathological tumor grade in neuroepithelial brain tumors by using spectral pattern analysis of in vivo spectroscopic data

OBJECT: In this study, 1H magnetic resonance (MR) spectroscopy was prospectively tested as a reliable method for presurgical grading of neuroepithelial brain tumors. METHODS: Using a database of tumor spectra obtained in patients with histologically confirmed diagnoses, 94 consecutive untreated patients were studied using single-voxel 1H spectroscopy (point-resolved spectroscopy; TE 135 msec, TE 135 msec, TR 1500 msec). A total of 90 tumor spectra obtained in patients with diagnostic 1H MR spectroscopy examinations were analyzed using commercially available software (MRUI/VARPRO) and classified using linear discriminant analysis as World Health Organization (WHO) Grade I/II, WHO Grade III, or WHO Grade IV lesions. In all cases, the classification results were matched with histopathological diagnoses that were made according to the WHO classification criteria after serial stereotactic biopsy procedures or open surgery. Histopathological studies revealed 30 Grade I/II tumors, 29 Grade III tumors, and 31 Grade IV tumors. The reliability of the histological diagnoses was validated considering a minimum postsurgical follow-up period of 12 months (range 12-37 months). Classifications based on spectroscopic data yielded 31 tumors in Grade I/II, 32 in Grade III, and 27 in Grade IV. Incorrect classifications included two Grade II tumors, one of which was identified as Grade III and one as Grade IV; two Grade III tumors identified as Grade II; two Grade III lesions identified as Grade IV; and six Grade IV tumors identified as Grade III. Furthermore, one glioblastoma (WHO Grade IV) was classified as WHO Grade I/II. This represents an overall success rate of 86%, and a 95% success rate in differentiating low-grade from high-grade tumors. CONCLUSIONS: The authors conclude that in vivo 1H MR spectroscopy is a reliable technique for grading neuroepithelial brain tumors.

Selective increases in regional brain glucocorticoid: a novel effect of chronic alcohol

The hypothalamo-pituitary-adrenal axis shows functional changes in alcoholics, with raised glucocorticoid release during alcohol intake and during the initial phase of alcohol withdrawal. Raised glucocorticoid concentrations are known to cause neuronal damage after withdrawal from chronic alcohol consumption and in other conditions. The hypothesis for these studies was that chronic alcohol treatment would have differential effects on corticosterone concentrations in plasma and in brain regions. Effects of chronic alcohol and withdrawal on regional brain corticosterone concentrations were examined using a range of standard chronic alcohol treatments in two strains of mice and in rats. Corticosterone was measured by radioimmunoassay and the identity of the corticosterone extracted from brain was verified by high performance liquid chromatography and mass spectrometry. Withdrawal from long term (3 weeks to 8 months) alcohol consumption induced prolonged increases in glucocorticoid concentrations in specific regions of rodent brain, while plasma concentrations remained unchanged. This effect was seen after alcohol administration via drinking fluid or by liquid diet, in both mice and rats and in both genders. Shorter alcohol treatments did not show the selective effect on brain glucocorticoid levels. During the alcohol consumption the regional brain corticosterone concentrations paralleled the plasma concentrations. Type II glucocorticoid receptor availability in prefrontal cortex was decreased after withdrawal from chronic alcohol consumption and nuclear localization of glucocorticoid receptors was increased, a pattern that would be predicted from enhanced glucocorticoid type II receptor activation. This novel observation of prolonged selective increases in brain glucocorticoid activity could explain important consequences of long term alcohol consumption, including memory loss, dependence and lack of hypothalamo-pituitary responsiveness. Local changes in brain glucocorticoid levels may also need to be considered in the genesis of other mental disorders and could form a potential new therapeutic target.

Wnt activation of immortalized brain endothelial cells as a tool for generating a standardized model of the blood brain barrier in vitro

Reproducing the characteristics and the functional responses of the blood-brain barrier (BBB) in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize β-catenin, or they are infected with a transcriptionally active form of β-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation.

Nonclassical mechanisms of progesterone action in the brain: I. Protein kinase C activation in the hypothalamus of female rats.

The modulation of gene regulation by progesterone (P) and its classical intracellular regulation by progestin receptors in the brain, resulting in alterations in physiology and behavior has been well studied. The mechanisms mediating the short latency effects of P are less well understood. Recent studies have revealed rapid nonclassical signaling action of P involving the activation of intracellular signaling pathways. We explored the involvement of protein kinase C (PKC) in P-induced rapid signaling in the ventromedial nucleus of the hypothalamus (VMN) and preoptic area (POA) of the rat brain. Both the Ca2+-independent (basal) PKC activity representing the activation of PKC by the in vivo treatments and the Ca+2-dependent (total) PKC activity assayed in the presence of exogenous cofactors in vitro were determined. A comparison of the two activities demonstrated the strength and temporal status of PKC regulation by steroid hormones in vivo. P treatment resulted in a rapid increase in basal PKC activity in the VMN but not the POA. Estradiol benzoate priming augmented P-initiated increase in PKC basal activity in both the VMN and POA. These increases were inhibited by intracerebroventricular administration of a PKC inhibitor administered 30 min prior to P. The total PKC activity remained unchanged demonstrating maximal PKC activation within 30 min in the VMN. In contrast, P regulation in the POA significantly attenuated total PKC activity +/- estradiol benzoate priming. These rapid changes in P-initiated PKC activity were not due to changes in PKC protein levels or phosphorylation status.

Brain rhythms alterations and their effect in functional networks: A simultaneous EEG/fMRI study in Fixation-off epilepsy

Fixation-off sensitivity (FOS) denotes the forms of EEG abnormalities, which are elicited by elimination of central vision or fixation. The phenomenon seems to depend on variables that modulate the alpha rhythm, however, the cerebral mechanisms underlying FOS remain unclear [1]. The scarce previous fMRI findings related to FOS have shown activation in extrastriate cortex [2] and also in frontal areas [3][4]. On the other hand, simultaneous EEG-fMRI technique has been used to assess the relationship between spontaneous power fluctuations of electrical rhythms and associated fMRI signal modulations. These studies have identified that lateral frontoparietal networks show a negative correlation with alpha band in healthy subjects. This neuroanatomical pattern is related to attentional processes and cognitive resources. Moreover, a sub-beta band (17-23 Hz) has been identified with posterior cingulate, temporoparietal junction and dorso-medial prefrontal cortex activations, which correspond to the DMN [5][6].

Repetitive transcranial magnetic stimulation activates specific regions in rat brain

Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive technique to induce electric currents in the brain. Although rTMS is being evaluated as a possible alternative to electroconvulsive therapy for the treatment of refractory depression, little is known about the pattern of activation induced in the brain by rTMS. We have compared immediate early gene expression in rat brain after rTMS and electroconvulsive stimulation, a well-established animal model for electroconvulsive therapy. Our result shows that rTMS applied in conditions effective in animal models of depression induces different patterns of immediate-early gene expression than does electroconvulsive stimulation. In particular, rTMS evokes strong neural responses in the paraventricular nucleus of the thalamus (PVT) and in other regions involved in the regulation of circadian rhythms. The response in PVT is independent of the orientation of the stimulation probe relative to the head. Part of this response is likely because of direct activation, as repetitive magnetic stimulation also activates PVT neurons in brain slices.

Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia

Ischemic stroke is the most common life-threatening neurological disease and has limited therapeutic options. One component of ischemic neuronal death is inflammation. Here we show that doxycycline and minocycline, which are broad-spectrum antibiotics and have antiinflammatory effects independent of their antimicrobial activity, protect hippocampal neurons against global ischemia in gerbils. Minocycline increased the survival of CA1 pyramidal neurons from 10.5% to 77% when the treatment was started 12 h before ischemia and to 71% when the treatment was started 30 min after ischemia. The survival with corresponding pre- and posttreatment with doxycycline was 57% and 47%, respectively. Minocycline prevented completely the ischemia-induced activation of microglia and the appearance of NADPH-diaphorase reactive cells, but did not affect induction of glial acidic fibrillary protein, a marker of astrogliosis. Minocycline treatment for 4 days resulted in a 70% reduction in mRNA induction of interleukin-1β-converting enzyme, a caspase that is induced in microglia after ischemia. Likewise, expression of inducible nitric oxide synthase mRNA was attenuated by 30% in minocycline-treated animals. Our results suggest that lipid-soluble tetracyclines, doxycycline and minocycline, inhibit inflammation and are neuroprotective against ischemic stroke, even when administered after the insult. Tetracycline derivatives may have a potential use also as antiischemic compounds in humans.

Cocaine-predictive stimulus induces drug-seeking behavior and neural activation in limbic brain regions after multiple months of abstinence: Reversal by D1 antagonists

The conditioning of cocaine's subjective actions with environmental stimuli may be a critical factor in long-lasting relapse risk associated with cocaine addiction. To study the significance of learning factors in persistent addictive behavior as well as the neurobiological basis of this phenomenon, rats were trained to associate discriminative stimuli (SD) with the availability of i.v. cocaine vs. nonrewarding saline solution, and then placed on extinction conditions during which the i.v. solutions and SDs were withheld. The effects of reexposure to the SD on the recovery of responding at the previously cocaine-paired lever and on Fos protein expression then were determined in two groups. One group was tested immediately after extinction, whereas rats in the second group were confined to their home cages for an additional 4 months before testing. In both groups, the cocaine SD, but not the non-reward SD, elicited strong recovery of responding and increased Fos immunoreactivity in the basolateral amygdala and medial prefrontal cortex (areas Cg1/Cg3). The response reinstatement and Fos expression induced by the cocaine SD were both reversed by selective dopamine D1 receptor antagonists. The undiminished efficacy of the cocaine SD to elicit drug-seeking behavior after 4 months of abstinence parallels the long-lasting nature of conditioned cue reactivity and cue-induced cocaine craving in humans, and confirms a significant role of learning factors in the long-lasting addictive potential of cocaine. Moreover, the results implicate D1-dependent neural mechanisms within the medial prefrontal cortex and basolateral amygdala as substrates for cocaine-seeking behavior elicited by cocaine-predictive environmental stimuli.

Functional analysis of retinoid Z receptor beta, a brain-specific nuclear orphan receptor.

The retinoid Z receptor beta (RZR beta), an orphan receptor, is a member of the retinoic acid receptor (RAR)/thyroid hormone receptor (TR) subfamily of nuclear receptors. RZR beta exhibits a highly restricted brain-specific expression pattern. So far, no natural RZR beta target gene has been identified and the physiological role of the receptor in transcriptional regulation remains to be elucidated. Electrophoretic mobility shift assays reveal binding of RZR beta to monomeric response elements containing the sequence AnnTAGGTCA, but RZR beta-mediated transactivation of reporter genes is only achieved with two property spaced binding sites. We present evidence that RZR beta can function as a cell-type-specific transactivator. In neuronal cells, GaI-RZR beta fusion proteins function as potent transcriptional activators, whereas no transactivation can be observed in nonneuronal cells. Mutational analyses demonstrate that the activation domain (AF-2) of RZR beta and RAR alpha are functionally interchangeable. However, in contrast to RAR and TR, the RZR beta AF-2 cannot function autonomously as a transactivation domain. Furthermore, our data define a novel repressor function for the C-terminal part of the putative ligand binding domain. We propose that the transcriptional activity of RZR beta is regulated by an interplay of different receptor domains with coactivators and corepressors.

Activation of single whisker barrel in rat brain localized by functional magnetic resonance imaging.

The previously established cortical representation of rat whiskers in layer IV of the cortex contains distinct cylindrical columns of cellular aggregates, which are termed barrels and correlate in a one-to-one relation to whiskers on the contralateral rat face. In the present study, functional magnetic resonance imaging (fMRI) of the rat brain was used to map whisker barrel activation during mechanical up-down movement (+/- 2.5 mm amplitude at 8 Hz) of single/multiple whisker(s). Multislice gradient echo fMRI experiments were performed at 7 T with in-plane image resolution of 220 x 220 microns, slice thickness of 1 mm, and echo time of 16 ms. Highly significant (P < 0.001) and localized contralateral regions of activation were observed upon stimulation of single/multiple whisker(s). In all experiments (n = 10), the locations of activation relative to bregma and midline were highly correlated with the neuroanatomical position of the corresponding whisker barrels, and the results were reproducible intra- and interanimal. Our results indicate that fMRI based on blood oxygenation level-dependent image contrast has the sensitivity to depict activation of a single whisker barrel in the rat brain. This noninvasive technique will supplement existing methods in the study of rat barrel cortex and should be particularly useful for the long-term investigations of central nervous system in the same animal.

Brain activity during automatic semantic priming revealed by event-related functional magnetic resonance imaging

Semantic priming occurs when a subject is faster in recognising a target word when it is preceded by a related word compared to an unrelated word. The effect is attributed to automatic or controlled processing mechanisms elicited by short or long interstimulus intervals (ISIs) between primes and targets. We employed event-related functional magnetic resonance imaging (fMRI) to investigate blood oxygen level dependent (BOLD) responses associated with automatic semantic priming using an experimental design identical to that used in standard behavioural priming tasks. Prime-target semantic strength was manipulated by using lexical ambiguity primes (e.g., bank) and target words related to dominant or subordinate meaning of the ambiguity. Subjects made speeded lexical decisions (word/nonword) on dominant related, subordinate related, and unrelated word pairs presented randomly with a short ISI. The major finding was a pattern of reduced activity in middle temporal and inferior prefrontal regions for dominant versus unrelated and subordinate versus unrelated comparisons, respectively. These findings are consistent with both a dual process model of semantic priming and recent repetition priming data that suggest that reductions in BOLD responses represent neural priming associated with automatic semantic activation and implicate the left middle temporal cortex and inferior prefrontal cortex in more automatic aspects of semantic processing.

Principal component analysis and assessment of language network activation patterns in pediatric epilepsy

This dissertation establishes a novel data-driven method to identify language network activation patterns in pediatric epilepsy through the use of the Principal Component Analysis (PCA) on functional magnetic resonance imaging (fMRI). A total of 122 subjects’ data sets from five different hospitals were included in the study through a web-based repository site designed here at FIU. Research was conducted to evaluate different classification and clustering techniques in identifying hidden activation patterns and their associations with meaningful clinical variables. The results were assessed through agreement analysis with the conventional methods of lateralization index (LI) and visual rating. What is unique in this approach is the new mechanism designed for projecting language network patterns in the PCA-based decisional space. Synthetic activation maps were randomly generated from real data sets to uniquely establish nonlinear decision functions (NDF) which are then used to classify any new fMRI activation map into typical or atypical. The best nonlinear classifier was obtained on a 4D space with a complexity (nonlinearity) degree of 7. Based on the significant association of language dominance and intensities with the top eigenvectors of the PCA decisional space, a new algorithm was deployed to delineate primary cluster members without intensity normalization. In this case, three distinct activations patterns (groups) were identified (averaged kappa with rating 0.65, with LI 0.76) and were characterized by the regions of: (1) the left inferior frontal Gyrus (IFG) and left superior temporal gyrus (STG), considered typical for the language task; (2) the IFG, left mesial frontal lobe, right cerebellum regions, representing a variant left dominant pattern by higher activation; and (3) the right homologues of the first pattern in Broca's and Wernicke's language areas. Interestingly, group 2 was found to reflect a different language compensation mechanism than reorganization. Its high intensity activation suggests a possible remote effect on the right hemisphere focus on traditionally left-lateralized functions. In retrospect, this data-driven method provides new insights into mechanisms for brain compensation/reorganization and neural plasticity in pediatric epilepsy.

Investigation of multisensory processing and structural brain differences in Autism Spectrum Disorder

This thesis is an investigation of structural brain abnormalities, as well as multisensory and unisensory processing deficits in autistic traits and Autism Spectrum Disorder (ASD). To achieve this, structural and functional magnetic resonance imaging (fMRI) and psychophysical techniques were employed. ASD is a neurodevelopmental condition which is characterised by the social communication and interaction deficits, as well as repetitive patterns of behaviour, interests and activities. These traits are thought to be present in a typical population. The Autism Spectrum Quotient questionnaire (AQ) was developed to assess the prevalence of autistic traits in the general population. Von dem Hagen et al. (2011) revealed a link between AQ with white matter (WM) and grey matter (GM) volume (using voxel-based-morphometry). However, their findings revealed no difference in GM in areas associated with social cognition. Cortical thickness (CT) measurements are known to be a more direct measure of cortical morphology than GM volume. Therefore, Chapter 2 investigated the relationship between AQ scores and CT in the same sample of participants. This study showed that AQ scores correlated with CT in the left temporo-occipital junction, left posterior cingulate, right precentral gyrus and bilateral precentral sulcus, in a typical population. These areas were previously associated with structural and functional differences in ASD. Thus the findings suggest, to some extent, autistic traits are reflected in brain structure - in the general population. The ability to integrate auditory and visual information is crucial to everyday life, and results are mixed regarding how ASD influences audiovisual integration. To investigate this question, Chapter 3 examined the Temporal Integration Window (TIW), which indicates how precisely sight and sound need to be temporally aligned so that a unitary audiovisual event can be perceived. 26 adult males with ASD and 26 age and IQ-matched typically developed males were presented with flash-beep (BF), point-light drummer, and face-voice (FV) displays with varying degrees of asynchrony and asked to make Synchrony Judgements (SJ) and Temporal Order Judgements (TOJ). Analysis of the data included fitting Gaussian functions as well as using an Independent Channels Model (ICM) to fit the data (Garcia-Perez & Alcala-Quintana, 2012). Gaussian curve fitting for SJs showed that the ASD group had a wider TIW, but for TOJ no group effect was found. The ICM supported these results and model parameters indicated that the wider TIW for SJs in the ASD group was not due to sensory processing at the unisensory level, but rather due to decreased temporal resolution at a decisional level of combining sensory information. Furthermore, when performing TOJ, the ICM revealed a smaller Point of Subjective Simultaneity (PSS; closer to physical synchrony) in the ASD group than in the TD group. Finding that audiovisual temporal processing is different in ASD encouraged us to investigate the neural correlates of multisensory as well as unisensory processing using functional magnetic resonance imaging fMRI. Therefore, Chapter 4 investigated audiovisual, auditory and visual processing in ASD of simple BF displays and complex, social FV displays. During a block design experiment, we measured the BOLD signal when 13 adults with ASD and 13 typically developed (TD) age-sex- and IQ- matched adults were presented with audiovisual, audio and visual information of BF and FV displays. Our analyses revealed that processing of audiovisual as well as unisensory auditory and visual stimulus conditions in both the BF and FV displays was associated with reduced activation in ASD. Audiovisual, auditory and visual conditions of FV stimuli revealed reduced activation in ASD in regions of the frontal cortex, while BF stimuli revealed reduced activation the lingual gyri. The inferior parietal gyrus revealed an interaction between stimulus sensory condition of BF stimuli and group. Conjunction analyses revealed smaller regions of the superior temporal cortex (STC) in ASD to be audiovisual sensitive. Against our predictions, the STC did not reveal any activation differences, per se, between the two groups. However, a superior frontal area was shown to be sensitive to audiovisual face-voice stimuli in the TD group, but not in the ASD group. Overall this study indicated differences in brain activity for audiovisual, auditory and visual processing of social and non-social stimuli in individuals with ASD compared to TD individuals. These results contrast previous behavioural findings, suggesting different audiovisual integration, yet intact auditory and visual processing in ASD. Our behavioural findings revealed audiovisual temporal processing deficits in ASD during SJ tasks, therefore we investigated the neural correlates of SJ in ASD and TD controls. Similar to Chapter 4, we used fMRI in Chapter 5 to investigate audiovisual temporal processing in ASD in the same participants as recruited in Chapter 4. BOLD signals were measured while the ASD and TD participants were asked to make SJ on audiovisual displays of different levels of asynchrony: the participants’ PSS, audio leading visual information (audio first), visual leading audio information (visual first). Whereas no effect of group was found with BF displays, increased putamen activation was observed in ASD participants compared to TD participants when making SJs on FV displays. Investigating SJ on audiovisual displays in the bilateral superior temporal gyrus (STG), an area involved in audiovisual integration (see Chapter 4), we found no group differences or interaction between group and levels of audiovisual asynchrony. The investigation of different levels of asynchrony revealed a complex pattern of results indicating a network of areas more involved in processing PSS than audio first and visual first, as well as areas responding differently to audio first compared to video first. These activation differences between audio first and video first in different brain areas are constant with the view that audio leading and visual leading stimuli are processed differently.