Connexin43 and astrocytic gap junctions in the rat spinal cord after acute compression injury.

To examine the possible role of interastrocytic gap junctions in the maintenance of tissue homeostasis after spinal cord damage, we initiated studies of the astrocytic gap junctional protein connexin43 (Cx43) in relation to temporal and spatial parameters of neuronal loss, reactive gliosis, and white matter survival in a rat model of traumatic spinal cord injury (SCI). Cx43 immunolocalization in normal and compression-injured spinal cord was compared by using two different sequence-specific anti-Cx43 antibodies that have previously exhibited different immunorecognition properties at lesion sites in brain. At 1- and 3-day survival times, gray matter areas with mild to moderate neuronal depletion exhibited a loss of immunolabeling with one of the two antibodies. At the lesion epicenter, these areas consisted of a zone that separated normal staining distal to the lesion from intensified labeling seen with both antibodies immediately adjacent to the lesion. Loss of immunoreactivity with only one of the two antibodies suggested masking of the corresponding Cx43 epitope. By 7 days post-SCI, Cx43 labeling was absent with both antibodies in all regions extending up to 1 mm from the lesion site. Reactive astrocytes displaying glial fibrillary acidic protein (GFAP) appeared by 1 day and were prominent by 3 days post-SCI. Their distribution in white and gray matter corresponded closely to that of Cx43 staining at 1 day, but less so at 3 days when GFAP-positive profiles were present at sites where Cx43 labeling was absent. By 7 days post-SCI, Cx43 again co-localized with GFAP-positive cells in the surviving subpial rim, and with astrocytic processes on radially oriented vascular profiles investing the central borders of the lesion. The results indicate that alterations in Cx43 cellular localization and Cx43 molecular modifications reflected by epitope masking, which were previously correlated with gap junction remodeling following excitotoxin-induced lesions in brain, are not responses limited to exogenously applied excitotoxins; they also occur in damaged spinal cord and are evoked by endogenous mechanisms after traumatic SCI. The GFAP/Cx43 co-localization results suggest that during their transformation to a reactive state, spinal cord astrocytes undergo a transitional phase marked by altered Cx43 localization or expression.

Connexin-43 in rat spinal cord: localization in astrocytes and identification of heterotypic astro-oligodendrocytic gap junctions.

Connexin-43 in relation to gap junctions between astrocytes and between other cell types in rat spinal cord was investigated immunohistochemically. In gray matter, connexin-43 was distributed thoughout all laminae, but was more concentrated in the substantia gelatinosa and around the central canal. Ultrastructurally, immunostaining was present in the cytoplasm of, and at gap junctions between, fine astrocytic processes, most of which ensheathed neuronal elements. In white matter, connexin-43 was localized to somata of fibrous astrocytes, their glial fibrillary acidic protein-positive processes running parallel to myelinated axons, and at gap junctions between these processes. Labelling was also evident in thick radially-directed astrocytic processes displaying pockets of staining near immunopositive gap junctions. Near the cord surface, staining was present in cell bodies of subpial astrocytes and at gap junctions between their tangential processes which formed most of the glia limitans. Radially-directed processes of subpial astrocytes formed symmetrically- and asymmetrically-labelled gap junctions with each other and extended fine branches into surrounding white matter where they made contact and often formed gap junctions with oligodendrocytic processes at the outer surface of myelinated fibres. Immunopositive astrocyte processes also made heterologous gap junctions with unstained oligodendrocyte cell bodies. Ependymal cells lining the central canal exhibited apical cytoplasmic labelling, as well as symmetrically-labelled gap junctions at their apices. Ependymal cells also formed asymmetrically-labelled gap junctions at which the junctional membranes of unlabelled cells, presumed to be tanycytes, were unstained. The results indicate the expression of connexins in addition to connexin-43 at asymmetrically-labelled gap junctions between some astrocytic processes, between astrocytes and oligodendrocytes and between some ependymal cells. The presence of gap junctions between astrocyte and oligodendrocyte processes at the outer surface of myelin suggests incorporation of the latter into the extensive gap junctionally-coupled astrocytic syncytium.

Functional magnetic resonance imaging of the human cervical spinal cord with stimulation of different sensory dermatomes.

Functional MR imaging (fMRI) of the cervical spinal cord was carried out in 13 healthy volunteers. A cold stimulus was applied, at different times, to three different sensory dermatome regions overlying the right hand and forearm: the thumb side of the palm, the little finger side of the palm, and the forearm below the elbow. Stimulation of these areas is expected to involve the 6(th), 8(th), and 5(th) cervical spinal cord segments respectively. Whereas true activations are expected to correspond to the region being stimulated, false activations such as arising from noise and motion, are not. The results demonstrate that clustering of active pixels into groups based on their intensity time courses discriminates false activations from true activations. Following clustering, the distribution of activity observed with fMRI matched the expected regions of neuronal activation with the different areas of stimulation on the hand and forearm.

Spin-echo versus gradient-echo fMRI with short echo times.

Blood-oxygen level dependent signal changes in the visual cortex were investigated as a function of echo time with spin-echo and gradient-echo EPI at 1.5 T and 3 T. The linear relationship between the fractional signal change and the echo time was apparent in all cases. Relaxation rate changes determined from the slope of this linear relation agree with published values, intercept values extrapolated to an echo time of zero, however, were 0.66% to 1.0% with spin-echo EPI, and 0.11% to 0.35% with gradient-echo EPI. Spin-echo and gradient-echo EPI can therefore yield similar signal changes at sufficiently short echo times.

Characterization of contrast changes in functional MRI of the human spinal cord at 1.5 T.

Contrast changes observed in functional magnetic resonance imaging in the human spinal cord were investigated with both motor and sensory tasks over a range of echo times. Data were acquired using a single-shot fast spin-echo sequence at 1.5 Tesla. Data were analyzed with two different correlation thresholds and the effects of altering the order of repeated experiments was also investigated. Plots of the fractional signal change as a function of echo time yielded linear functions with slopes corresponding to relaxation rate changes of -0.30 sec(-1) with sensory stimulation and approximately -0.50 sec(-1) with a motor task. However, the fractional signal change extrapolated to an echo time of zero was significantly greater than zero in each case and was roughly 2.5%. This suggests that in addition to the BOLD effect there is a baseline signal change which occurs concomitant to neuronal activation in the spinal cord.

Functional magnetic resonance imaging of the human brain based on signal enhancement by extravascular protons (SEEP fMRI).

Functional magnetic resonance imaging (fMRI) studies of the human brain were carried out at 3 Tesla to investigate an fMRI contrast mechanism that does not arise from the blood oxygen-level dependent (BOLD) effect. This contrast mechanism, signal enhancement by extravascular protons (SEEP), involves only proton-density changes and was recently demonstrated to contribute to fMRI signal changes in the spinal cord. In the present study it is hypothesized that SEEP fMRI can be used to identify areas of neuronal activity in the brain with as much sensitivity and precision as can be achieved with BOLD fMRI. A detailed analysis of the areas of activity, signal intensity time courses, and the contrast-to-noise ratio (CNR), is also presented and compared with the BOLD fMRI results. Experiments were carried out with subjects performing a simple finger-touching task, or observing an alternating checkerboard pattern. Data were acquired using a conventional BOLD fMRI method (gradient-echo (GE) EPI, TE = 30 ms), a conventional method with reduced BOLD sensitivity (GE-EPI, TE = 12 ms), and SEEP fMRI (spin-echo (SE) EPI, TE = 22 ms). The results of this study demonstrate that SEEP fMRI may provide better spatial localization of areas of neuronal activity, and a higher CNR than conventional BOLD fMRI, and has the added benefit of lower sensitivity to field inhomogeneities.

Mapping of neuronal function in the healthy and injured human spinal cord with spinal fMRI.

Functional magnetic resonance imaging of the human spinal cord is carried out with a graded thermal stimulus in order to establish the relationship between signal changes and neural activity. Studies of the lumbar spinal cord in 15 healthy subjects with 10 degrees C stimulation of the skin overlying the calf demonstrate a pattern of activity that matches the neuronal anatomy of the spinal cord. This pattern shows primarily dorsal horn activity, with expected components of motor reflex activity as well. Moreover, a later response shifting to noxious cold over time is also demonstrated with a shift to more dorsal horn activity. Signal intensity changes detected at different degrees of thermal stimulation have a biphasic nature, with much larger signal changes below 15 degrees C as the stimulus becomes noxious, and agree well with electrophysiological results reported in the literature. These findings demonstrate a strong correspondence between Spinal fMRI results and neural activity in the human spinal cord. Spinal fMRI is also applied to studies of the injured spinal cord, below the site of injury. Results consistently demonstrate activity in the spinal cord even when the subjects cannot feel the stimulus being applied. Signal intensity changes demonstrate the same stimulus-response pattern as that in noninjured subjects, but the areas of activity in the spinal gray matter are notably altered. In subjects with complete injuries, activity is absent ipsilateral to the thermal stimulation, but appears to be enhanced on the contralateral side. These findings demonstrate the reliability of Spinal fMRI and its clinical potential.

Requirement of the hyaluronan receptor RHAMM in neurite extension and motility as demonstrated in primary neurons and neuronal cell lines.

The recently cloned and characterized hyaluronan (HA) receptor RHAMM (receptor for HA-mediated motility) has been shown to play a critical role in mechanisms underlying the motile capacity of a variety of peripheral cell types. Similarities in molecular processes that govern cell locomotion and growth cone migration prompted us to investigate whether RHAMM also contributes to neurite migration in vitro. In immunohistochemical studies of PC12 cells, NG108-15 cells and a neuroblastoma/spinal cord neuronal hybrid cell line (NSC-34 cells) as well as rat and human primary neurons, a punctiform RHAMM labeling pattern was detected in cell bodies, along processes, and at growth cones. By Western blot analysis, the cells lines expressed major RHAMM forms with apparent MW of 60, 75, and 116 kDa. Treatment of NG108-15 cells with dibutyryl-cAMP led to a clear increase in immunolabeling for RHAMM and enhanced expression of the 60 and 75 kDa forms. A polyclonal anti-RHAMM antibody that interferes with HA/RHAMM interaction significantly reduced neurite migration of each cell type examined, while another directed against a RHAMM repeat sequence thought to promote RHAMM receptor aggregation significantly stimulated neurite migration of NSC-34 and rat primary neurons. Different monoclonal anti-RHAMM antibodies had differential inhibitory actions on neurite movement. Low concentrations (ng/ml) of a peptide corresponding to an HA binding domain within RHAMM inhibited neurite migration. These results are the first to implicate RHAMM in the mediation of neurite motility and migration and to point to the potential importance of HA in this process.

Distraction modulates anterior cingulate gyrus activations during the cold pressor test.

The anterior cingulate gyrus (ACG) is part of a neural network implicated in attention-demanding tasks, such as the experience of pain. However, the regions within the ACG responding to cognitive demands and to painful stimulation are not identical. Since directing attention away from a painful stimulus is known to reduce the perceived pain intensity, we hypothesized that distraction from pain would result both in decreased activation of ACG subregions responsive to painful stimulation and increased activation of ACG subregions responsive to the distraction task. BOLD fMRI has comparatively high spatial resolution and allows for better identification of ACG subregional responses than other neuroimaging techniques. Twelve subjects were tested using the cold pressor test (CPT), a verbal attention task (VAT), and a distraction task (DT) (a combination of the CPT and VAT). Analysis was performed on a voxel-by-voxel basis using a general linear model as implemented in SPM99. In addition to ACG activations common to both the CPT and VAT, we identified one CPT-specific cluster in an area corresponding to BA24'. The modulation effect of distraction on pain was assessed by contrasting (CPT-DT) and (DT-CPT). In support of our hypothesis, contrast (CPT-DT) revealed a decrease in BA24' during the DT and contrast (DT-CPT) showed increased activation in BA32/32'. These data suggest that distraction from pain and concomitant low pain ratings are reflected in distinct ACG subregional responses.

Extravascular proton-density changes as a non-BOLD component of contrast in fMRI of the human spinal cord.

The fractional signal intensity change (Delta S/S) observed during activation in T(2)-weighted fMRI of the spinal cord has previously been shown to depend linearly on the echo time (TE) but to have a positive value of roughly 2.5% extrapolated to zero TE. In this study we investigated the origin of this finding by measuring the Delta S/S in spinal fMRI with very short TEs. Our results demonstrate that the Delta S/S does not approach zero, but has a value as high as 3.3% at TE = 11 ms. At TEs > 33 ms we observed the linear relationship between Delta S/S and TE as in previous studies. These data demonstrate that there is a non-BOLD contribution to signal changes observed in spinal fMRI. We hypothesize that this contribution is a local proton density increase due to increased water exudation from capillaries with increased blood flow during neuronal activation, and term this effect "signal enhancement by extravascular protons" (SEEP).