Christoph Hofstetter

Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery

Marrow stromal cells (MSC) can be expanded rapidly in vitro and differentiated into multiple mesodermal cell types. In addition, differentiation into neuron-like cells expressing markers typical for mature neurons has been reported. To analyze whether such cells, exposed to differentiation media, could develop electrophysiological properties characteristic of neurons, we performed whole-cell recordings. Neuron-like MSC, however, lacked voltage-gated ion channels necessary for generation of action potentials. We then delivered MSC into the injured spinal cord to study the fate of transplanted MSC and possible effects on functional outcome in animals rendered paraplegic. MSC given 1 week after injury led to significantly larger numbers of surviving cells than immediate treatment and significant improvements of gait. Histology 5 weeks after spinal cord injury revealed that MSC were tightly associated with longitudinally arranged immature astrocytes and formed bundles bridging the epicenter of the injury. Robust bundles of neurofilament-positive fibers and some 5-hydroxytryptamine-positive fibers were found mainly at the interface between graft and scar tissue. MSC constitute an easily accessible, easily expandable source of cells that may prove useful in the establishment of spinal cord repair protocols.

Allodynia limits the usefulness of intraspinal neural stem cell grafts; directed differentiation improves outcome

Several studies have reported functional improvement after transplantation of neural stem cells into injured spinal cord. We now provide evidence that grafting of adult neural stem cells into a rat thoracic spinal cord weight-drop injury improves motor recovery but also causes aberrant axonal sprouting associated with allodynia-like hypersensitivity of forepaws. Transduction of neural stem cells with neurogenin-2 before transplantation suppressed astrocytic differentiation of engrafted cells and prevented graft-induced sprouting and allodynia. Transduction with neurogenin-2 also improved the positive effects of engrafted stem cells, including increased amounts of myelin in the injured area, recovery of hindlimb locomotor function and hindlimb sensory responses, as determined by functional magnetic resonance imaging. These findings show that stem cell transplantation into injured spinal cord can cause severe side effects and call for caution in the consideration of clinical trials.

Progressive parkinsonism in mice with respiratory-chain-deficient dopamine neurons

Mitochondrial dysfunction is implicated in the pathophysiology of Parkinson′s disease (PD), a common age-associated neurodegenerative disease characterized by intraneuronal inclusions (Lewy bodies) and progressive degeneration of the nigrostriatal dopamine (DA) system. It has recently been demonstrated that midbrain DA neurons of PD patients and elderly humans contain high levels of somatic mtDNA mutations, which may impair respiratory chain function. However, clinical studies have not established whether the respiratory chain deficiency is a primary abnormality leading to inclusion formation and DA neuron death, or whether generalized metabolic abnormalities within the degenerating DA neurons cause secondary damage to mitochondria. We have used a reverse genetic approach to investigate this question and created conditional knockout mice (termed MitoPark mice), with disruption of the gene for mitochondrial transcription factor A (Tfam) in DA neurons. The knockout mice have reduced mtDNA expression and respiratory chain deficiency in midbrain DA neurons, which, in turn, leads to a parkinsonism phenotype with adult onset of slowly progressive impairment of motor function accompanied by formation of intraneuronal inclusions and dopamine nerve cell death. Confocal and electron microscopy show that the inclusions contain both mitochondrial protein and membrane components. These experiments demonstrate that respiratory chain dysfunction in DA neurons may be of pathophysiological importance in PD.

Vascular endothelial growth factor improves functional outcome and decreases secondary degeneration in experimental spinal cord contusion injury.

Spinal cord injury leads to acute local ischemia, which may contribute to secondary degeneration. Hypoxia stimulates angiogenesis through a cascade of events, involving angiogenesis stimulatory substances, such as vascular endothelial growth factor (VEGF). To test the importance of angiogenesis for functional outcome and wound healing in spinal cord injury VEGF165 (proangiogenic), Ringers (control) or angiostatin (antiangiogenic) were delivered locally immediately after a contusion injury produced using the NYU impactor and a 25 mm weight-drop. Rats treated with VEGF showed significantly improved behavior up to 6 weeks after injury compared with control animals, while angiostatin treatment lead to no statistically significant changes in behavior outcome. Furthermore, VEGF-treated animals had an increased amount of spared tissue in the lesion center and a higher blood vessel density in parts of the wound area compared with controls. These effects were unlikely to be due to increased cell proliferation as determined by bromo-deoxy-uridine-labeling. Moreover, VEGF treatment led to decreased levels of apoptosis, as revealed by TUNEL assays. In situ hybridization demonstrated presence of mRNA for VEGF receptors Flt-1, fetal liver kinase-1, neuropilin-1 and -2 in several important cellular compartments of the spinal cord. The different experiments indicate that beneficial effects seen by acute VEGF delivery was attributable to protection/repair of blood vessels, decreased apoptosis and possibly also by other additional effects on glial cells or certain neuron populations.

In vivo magnetic resonance tracking of olfactory ensheathing glia grafted into the rat spinal cord.

Engraftment of olfactory ensheathing cells (OEC), a unique type of glia required for olfactory nerve growth throughout life, has been shown to foster axonal regeneration in different types of CNS and PNS injuries. However, a lack of suitable markers of OEC has hindered studies assessing survival and function of OEC grafts following transplantation. The aim of this study was to examine the possible usefulness of superparamagnetic iron oxide nanoparticles (magnetodendrimers) as a label to allow in vivo tracking of grafted OEC by MR imaging and to determine temporal and spatial migration of OEC in normal and injured rat spinal cords, including the possibility of such cells to cross a complete spinal cord injury zone. We found that labeled OEC were readily detectable in vivo by MR imaging for at least 2 months. Labeled OEC migrated extensively in normal spinal cord as shown by MRI and histological markers. In contrast, OEC showed limited migration in transected spinal cord and were not able to cross the transection gap. Furthermore, iron-containing hemorrhage products confounded interpretation of MR contrast patterns in the injured spinal cord. We conclude that (1) MR imaging is useful for noninvasive observation of cell migration dynamics after grafting in vivo, although interpretation in severe injuries should be cautious, and that (2) OEC migratory and thus regeneration-enhancing ability is limited when confronted with the glial scar of a transected spinal cord.

Blood Oxygenation Level-Dependent Visualization of Synaptic Relay Stations of Sensory Pathways along the Neuroaxis in Response to Graded Sensory Stimulation of a Limb

Blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) was used to test at which levels of the neuroaxis signals are elicited when different modalities of sensory information from the limbs ascend to cortex cerebri. We applied graded electric stimuli to the rat hindlimbs and used echo-planar imaging to monitor activity changes in the lumbar spinal cord and medulla oblongata, where primary afferents of painful and nonpainful sensation synapse, respectively. BOLD signals were detected in ipsilateral lumbar spinal cord gray matter using sufficiently strong stimuli. Using stimuli well below the threshold needed for signals to be elicited in the spinal cord, we found BOLD responses in dorsal medulla oblongata. The distribution of these signals is compatible with the neuroanatomy of the respective synaptic relay stations of the corresponding sensory pathways. Hence, the sensory pathways conducting painful and nonpainful information were successfully distinguished. The fMRI signals in the spinal cord were markedly decreased by morphine, and these effects were counteracted by naloxone. We conclude that fMRI can be used as a reliable and valid method to monitor neuronal activity in the rat spinal cord and medulla oblongata in response to sensory stimuli. Previously, we also documented BOLD signals from thalamus and cortex. Thus, BOLD responses can be elicited at all principal synaptic relay stations along the neuroaxis from lumbar spinal cord to sensory cortex. Rat spinal cord fMRI should become a useful tool in experimental spinal cord injury and pain research.

Moderate ethanol consumption increases hippocampal cell proliferation and neurogenesis in the adult mouse

Alcoholism is a lifelong disease often associated with emotional disturbances and a high risk of relapse even years after detoxification. To explore if cell proliferation in the dentate gyrus of the hippocampus might be important for alcohol-induced brain adaptation, we analysed hippocampal neurogenesis and gliogenesis in adult C57BL/6 mice that consumed moderate levels of ethanol (~6 g/kg.d) in a two-bottle free-choice model during ~10 wk. The mice developed a 53% preference for ethanol vs. water and displayed a blood ethanol concentration of 0.24 per thousand at the time of sacrifice. Bromo-deoxy-uridine (BrdU) was administered in different regimes to analyse proliferation, survival, cell distribution and differentiation of new cells in the dentate gyrus. Moderate ethanol consumption increased the proliferation of cells, which survived and developed a neural phenotype. Ethanol consumption did not induce apoptosis, neither did it change differentiation or the distribution patterns of the newly formed cells. The cell proliferation rate in the dentate gyrus returned to basal levels 3 d after ethanol withdrawal. We conclude that voluntary ethanol intake by mice can change the rate of cell proliferation in the dentate gyrus. These observations add to the emerging picture of dentate gyrus neurogenesis as a highly regulated process. Since there was no increase in apoptosis concomitant with the ethanol-induced increase in neurogenesis, it is possible that the new cells in the dentate gyrus may contribute to the long-lasting changes of brain function after ethanol consumption.

RhoA, RhoB, RhoC, Rac1, Cdc42, and Tc10 mRNA levels in spinal cord, sensory ganglia, and corticospinal tract neurons and long-lasting specific changes following spinal cord injury.

Inhibition of RhoA has been shown to enhance axonal regeneration following spinal cord injury. Here we mapped mRNA expression patterns of RhoA, B, and C, Rac1, Cdc42, and Tc10 in spinal cord, sensory ganglia, and sensorimotor cortex in uninjured rats, and following spinal cord injury or sham laminectomy. In the intact spinal cord, neurons displayed high levels of Rac1, Cdc42, and Tc10 mRNA hybridization signal. GFAP‐immunoreactive astrocytes expressed primarily RhoB and Rac1, while oligodendrocyte‐like cells expressed RhoA, Rac1, and Cdc42. Injury caused profound, long‐lasting upregulation of RhoA, Rac1, Cdc42, and Tc10 mRNA in the spinal cord, while RhoB was modestly increased and RhoC did not change. GFAP‐immunoreactive reactive astrocytes exhibited a dramatic increase of RhoA mRNA expression along with increases of Rac1 and Cdc42. Injury also led to elevation of RhoA, Cdc42, and Tc10 in neurons and modest increases of RhoA, Rac1, and Tc10 in oligodendrocyte‐like cells. Laminectomy caused similar, but less pronounced alterations of investigated mRNA species. In dorsal root ganglia neuronal RhoA, Rac1, Cdc42, and Tc10 mRNA levels were increased similarly by spinal cord injury and sham surgery. The CST pyramidal cells expressed Tc10 mRNA and the CST itself was Tc10‐immunoreactive. Tc10‐immunoreactivity disappeared distal to injury. We conclude that there are gene‐specific patterns of expression of the six different Rho‐GTPases in normal spinal cord and dorsal root ganglia, and that specific changes of temporal and spatial expression patterns occur in response to spinal cord injury, suggesting different roles of these GTPases in the cellular sequelae of CNS injury. J. Comp. Neurol. 484:224–233, 2005.

Numb rats walk: a behavioural and fMRI comparison of mild and moderate spinal cord injury

Assessment of sensory function serves as a sensitive measure for predicting the functional outcome following spinal cord injury in patients. However, little is known about loss and recovery of sensory function in rodent spinal cord injury models as most tests of sensory functions rely on behaviour and thus motor function. We used functional magnetic resonance imaging (fMRI) to investigate cortical and thalamic BOLD‐signal changes in response to limb stimulation following mild or moderate thoracic spinal cord weight drop injury in Sprague–Dawley rats. While there was recovery of close to normal hindlimb motor function as determined by open field locomotor testing following both degrees of injury, recovery of hindlimb sensory function as determined by fMRI and hotplate testing was only seen following mild injury and not following moderate injury. Thus, moderate injury can lead to near normal hindlimb motor function in animals with major sensory deficits. Recovered fMRI signals following mild injury had a partly altered cortical distribution engaging also ipsilateral somatosensory cortex and the cingulate gyrus. Importantly, thoracic spinal cord injury also affected sensory representation of the upper nonaffected limbs. Thus, cortical and thalamic activation in response to forelimb stimulation was significantly increased 16 weeks after spinal cord injury compared to control animals. We conclude that both forelimb and hindlimb cortical sensory representation is altered following thoracic spinal cord injury. Furthermore tests of sensory function that are independent of motor behaviour are needed in rodent spinal cord injury research.

The neuropeptide tyrosine Y1R is expressed in interneurons and projection neurons in the dorsal horn and area X of the rat spinal cord.

The localization of the neuropeptide tyrosine Y1 receptor was studied with immunohistochemistry in parasagittal and transverse, free-floating sections of the rat lumbar spinal cord. At least seven distinct Y1 receptor-positive populations could tentatively be recognized: Type 1) abundant small, fusiform Y1 receptor-positive neurons in laminae I-II, producing a profuse neuropil; Type 2) Y1 receptor-positive projection neurons in lamina I; Type 3) small Y1 receptor-positive neurons in lamina III, similar to Type 1 neurons, but less densely packed; Type 4) a number of large, multipolar Y1 receptor-positive neurons in the border area between laminae III-IV, with dendrites projecting toward laminae I-II; Type 5) a considerable number of large, multipolar Y1 receptor-positive neurons in laminae V-VI; Type 6) many large Y1 receptor-positive neurons around the central canal (area X); and Type 7) a small number of large Y1 receptor-positive neurons in the medial aspect of the ventral horns (lamina VIII). Many of the neurons present in laminae V-VI and area X produce craniocaudal processes extending for several hundred micrometers. Retrograde tracing using cholera toxin B subunit injected at the 9th thoracic spinal cord level shows that several Type 5 neurons in laminae V-VI, and at least a few Type 2 in lamina I and Type 6 in area X have projections extending to the lower segments of the thoracic spinal cord (and perhaps to supraspinal levels). The present results define distinct subpopulations of neuropeptide tyrosine-sensitive neurons, localized in superficial and deep layers of the dorsal, in the ventral horns and in area X. The lamina II neurons express somatostatin [The neuropeptide Y Y1 receptor is a somatic receptor on dorsal root ganglion neurons and a postsynaptic receptor on somatostatin dorsal horn neurons. Eur J Neurosci 11:2211-2225] and are presumably glutamatergic [Todd AJ, Hughes DI, Polgar E, Nagy GG, Mackie M, Ottersen OP, Maxwell DJ (2003) The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal horn. Eur J Neurosci 17:13-27], that is they are excitatory interneurons under a Y1 receptor-mediated inhibitory influence. The remaining Y1 receptor-positive spinal neurons need to be phenotyped, for example if the large Y1 receptor-positive laminae III-IV neurons (Type 5) are identical to the neurokinin (NK)1R-positive neurons previously shown to receive neuropeptide tyrosine positive dendritic contacts [Polgár E, Shehab SA, Watt C, Todd AJ (1999) GABAergic neurons that contain neuropeptide Y selectively target cells with the NK1 receptor in laminae III and IV of the rat spinal cord. J Neurosci 19:2637-2646]. If so, neuropeptide tyrosine could have an antinociceptive action not only via Y1 receptor-positive interneurons (Type 1) but also projection neurons. The present results show neuropeptide tyrosine-sensitive neuron populations virtually in all parts of the lumbar spinal cord, suggesting a role for neuropeptide tyrosine signaling in many spinal functions, including pain.