41 research outputs found
Microelectrode arrays in combination with in vitro models of spinal cord injury as tools to investigate pathological changes in network activity: facts and promises
No full textMicroelectrode arrays (MEAs) represent an important tool to study the basic characteristics of spinal networks that control locomotion in physiological conditions. Fundamental properties of this neuronal rhythmicity like burst origin, propagation, coordination and resilience can, thus, be investigated at multiple sites within a certain spinal topography and neighbouring circuits. A novel challenge will be to apply this technology to unveil the mechanisms underlying pathological processes evoked by spinal cord injury. To achieve this goal, it is necessary to fully identify spinal networks that make up the locomotor central pattern generator (CPG) and to understand their operational rules. In this review, the use of isolated spinal cord preparations from rodents, or organotypic spinal slice cultures is discussed to study rhythmic activity. In particular, this review surveys our recently developed in vitro models of spinal cord injury by evoking excitotoxic (or even hypoxic/dysmetabolic) damage to spinal networks and assessing their impact on rhythmic activity and cell survival. These pathological processes which evolve via different cell death mechanisms are discussed as a paradigm to apply MEA recording for detailed mapping of the functional damage and its time-dependent evolution. © 2013 Mladinic and Nistri
Dynamic expression of ATF3 as a novel tool to study activation and migration of endogenous spinal stem cells and their role in neural repair
No full textThe differential intracellular expression of the novel marker ATF-3 characterizes the quiescent or activated state of endogenous spinal stem cells: a tool to study neurorepair?
No full textWorldwide, spinal cord injury (SCI) remains a major cause of disability with serious consequences in terms of personal and social costs [1]. Thus, important issues are how to protect the spinal cord to limit its initial damage, how to repair a lesion, and how to facilitate recovery by exploiting surviving tissue. These needs are currently unmet because our knowledge of the detailed structure of the neuronal networks responsible for human locomotion is scanty and our control over the mechanisms involved in neuronal death and regeneration is very limited. The molecular mechanisms underlying neuronal death after SCI are incompletely understood so that specific strategies for neuroprotection remain preliminary [2-4]. While many neuroprotective molecules have been reported to be experimentally effective for neuronal survival after SCI, very few have reached the clinical testing stage and none of them has provided efficacious treatment for SCI patients [5]. The reasons for such a clinical failure are complex and may include the diversity of protocols used to induce injury in animal models and the difficulty of detailed animal tissue analysis beyond a single time point so that a relatively narrow window of pathophysiology may be explored [6,7]. In clinical settings, the large majority of SCI cases are managed at late stages after the patient’s conditions have been stabilized following the primary lesion. Hence, damage repair rather than neuroprotection becomes a crucial goal
Mechanisms underlying cell death in ischemia-like damage to the rat spinal cord in vitro
No full textNew spinal cord injury (SCI) cases are frequently due to non-traumatic causes, including vascular disorders. To develop mechanism-based neuroprotective strategies for acute SCI requires full understanding of the early pathophysiological changes to prevent disability and paralysis. The aim of our study was to identify the molecular and cellular mechanisms of cell death triggered by a pathological medium (PM) mimicking ischemia in the rat spinal cord in vitro. We previously showed that extracellular Mg2+ (1 mM) worsened PM-induced damage and inhibited locomotor function. The present study indicated that 1 h of PM+Mg2+ application induced delayed pyknosis chiefly in the spinal white matter via overactivation of poly (ADP-ribose) polymerase 1 (PARP1), suggesting cell death mediated by the process of parthanatos that was largely suppressed by pharmacological block of PARP-1. Gray matter damage was less intense and concentrated in dorsal horn neurons and motoneurons that became immunoreactive for the mitochondrial apoptosis-inducing factor (the intracellular effector of parthanatos) translocated into the nucleus to induce chromatin condensation and DNA fragmentation. Immunoreactivity to TRPM ion channels believed to be involved in ischemic brain damage was also investigated. TRPM2 channel expression was enhanced 24 h later in dorsal horn and motoneurons, whereas TRPM7 channel expression concomitantly decreased. Conversely, TRPM7 expression was found earlier (3 h) in white matter cells, whereas TRPM2 remained undetectable. Simulating acute ischemic-like damage in vitro in the presence of Mg2+ showed how, during the first 24 h, this divalent cation unveiled differential vulnerability of white matter cells and motoneurons, with distinct changes in their TRPM expression. © 2013 Macmillan Publishers Limited All rights reserved
Excitotoxic cell death induces delayed proliferation of endogenous neuroprogenitor cells in organotypic slice cultures of the rat spinal cord
No full textThe aim of the present report was to investigate whether, in the mammalian spinal cord, cell death induced by transient excitotoxic stress could trigger activation and proliferation of endogenous neuroprogenitor cells as a potential source of a lesion repair process and the underlying time course. Because it is difficult to address these issues in vivo, we used a validated model of spinal injury based on rat organotypic slice cultures that retain the fundamental tissue cytoarchitecture and replicate the main characteristics of experimental damage to the whole spinal cord. Excitotoxicity evoked by 1 h kainate application produced delayed neuronal death (40%) peaking after 1 day without further losses or destruction of white matter cells for up to 2 weeks. After 10 days, cultures released a significantly larger concentration of endogenous glutamate, suggesting functional network plasticity. Indeed, after 1 week the total number of cells had returned to untreated control level, indicating substantial cell proliferation. Activation of progenitor cells started early as they spread outside the central area, and persisted for 2 weeks. Although expression of the neuronal progenitor phenotype was observed at day 3, peaked at 1 week and tapered off at 2 weeks, very few cells matured to neurons. Astroglia precursors started proliferating later and matured at 2 weeks. These data show insult-related proliferation of endogenous spinal neuroprogenitors over a relatively brief time course, and delineate a narrow temporal window for future experimental attempts to drive neuronal maturation and for identifying the factors regulating this process. © 2013 Macmillan Publishers Limited. All rights reserved
Dynamics of early locomotor network dysfunction following a focal lesion in an in vitro model of spinal injury
No full textIt is unclear how a localized spinal cord injury may acutely affect locomotor networks of segments initially spared by the lesion. To
investigate the process of secondary damage following spinal injury, we used the in vitro model of the neonatal rat isolated spinal cord
with transverse barriers at the low thoracic–upper lumbar region to allow focal application of kainate in hypoxic and aglycemic solution
(with reactive oxygen species). The time-course and nature of changes in spinal locomotor networks downstream of the lesion site
were investigated over the first 24 h, with electrophysiological recordings monitoring fictive locomotion (alternating oscillations
between flexor and extensor motor pools on either side) and correlating any deficit with histological alterations. The toxic solution
irreversibly suppressed synaptic transmission within barriers without blocking spinal reflexes outside. This effect was focally
associated with extensive white matter damage and ventral gray neuronal loss. Although cell losses were < 10% outside barriers,
microglial activation with neuronal phagocytosis was detected. Downstream motor networks still generated locomotor activity 24 h
later when stimulated with N-methyl-d-aspartate (NMDA) and serotonin, but not with repeated dorsal root stimuli. In the latter case,
cumulative depolarization was recorded from ventral roots at a slower rate of rise, suggesting failure to recruit network
premotoneurons. Our data indicate that, within the first 24 h of injury, locomotor networks below the lesion remained morphologically
intact and functional when stimulated by NMDA and serotonin. Nevertheless, microglial activation and inability to produce locomotor
patterns by dorsal afferent stimuli suggest important challenges to long-term network operation
Deconstructing locomotor networks with experimental injury to define their membership
No full textAlthough spinal injury is a major cause of chronic disability, the mechanisms responsible for the lesion pathophysiology
and their dynamic evolution remain poorly understood. Hence, current treatments aimed at blocking damage
extension are unsatisfactory. To unravel the acute spinal injury processes, we have developed a model of the neonatal
rat spinal cord in vitro subjected to kainate-evoked excitotoxicity ormetabolic perturbation (hypoxia, aglycemia, and
free oxygen radicals) or their combination. The study outcome is fictive locomotion one day after the lesion and its
relation to histological damage. Excitotoxicity always suppresses locomotor network activity and produces large gray
matter damage, while network bursting persists supported by average survival of nearly half premotoneurons and
motoneurons. Conversely, metabolic perturbation simply depresses locomotor network activity as damage mainly
concerns white rather than gray matter. Coapplication of kainate and metabolic perturbation completely eliminates
locomotor network activity. These results indicate distinct cellular targets for excitotoxic versus dysmetabolic damage
with differential consequences on locomotor pattern formation. Furthermore, these data enable to estimate the
minimal network membership compatible with expression of locomotor activity
Kainate and metabolic perturbation mimicking spinal injury differentially contribute to early damage of locomotor networks in the in vitro neonatal rat spinal cord
No full textAcute spinal cord injury evolves rapidly to produce
secondary damage even to initially spared areas. The
result is loss of locomotion, rarely reversible in man. It is,
therefore, important to understand the early pathophysiological
processes which affect spinal locomotor networks. Regardless
of their etiology, spinal lesions are believed to include
combinatorial effects of excitotoxicity and severe
stroke-like metabolic perturbations. To clarify the relative
contribution by excitotoxicity and toxic metabolites to dysfunction
of locomotor networks, spinal reflexes and intrinsic
network rhythmicity, we used, as a model, the in vitro thoraco-
lumbar spinal cord of the neonatal rat treated (1 h) with
either kainate or a pathological medium (containing free radicals
and hypoxic/aglycemic conditions), or their combination.
After washout, electrophysiological responses were
monitored for 24 h and cell damage analyzed histologically.
Kainate suppressed fictive locomotion irreversibly, while it
reversibly blocked neuronal excitability and intrinsic bursting
induced by synaptic inhibition block. This result was associated
with significant neuronal loss around the central canal.
Combining kainate with the pathological medium evoked extensive,
irreversible damage to the spinal cord. The pathological
medium alone slowed down fictive locomotion and
intrinsic bursting: these oscillatory patterns remained
throughout without regaining their control properties. This
phenomenon was associated with polysynaptic reflex depression
and preferential damage to glial cells, while neurons
were comparatively spared. Our model suggests distinct
roles of excitotoxicity and metabolic dysfunction in the acute
damage of locomotor networks, indicating that different strategies
might be necessary to treat the various early components
of acute spinal cord lesion
Postnatal developmental profile of neurons and glia in motor nuclei of the brainstem and spinal cord, and its comparison with organotypic slice cultures
No full textIn vitro preparations of the neonatal rat spinal cord or brainstem are useful to investigate the organization of motor networks and their dysfunction in neurological disease models. Long-term spinal cord organotypic cultures can extend our understanding of such pathophysiological processes over longer times. It is, however, surprising that detailed descriptions of the type (and number) of neurons and glia in such preparations are currently unavailable to evaluate cell-selectivity of experimental damage. The focus of the present immunohistochemical study is the novel characterization of the cell population in the lumbar locomotor region of the rat spinal cord and in the brainstem motor nucleus hypoglossus at 04 postnatal days, and its comparison with spinal organotypic cultures at 222 days in vitro. In the nucleus hypoglossus, neurons were 40% of all cells and 80% of these were motoneurons. Astrocytes (35% of total cells) were the main glial cells, while microglia was <10%. In the spinal gray matter, the highest neuronal density was in the dorsal horn (>80%) and the lowest in the ventral horn (=57%) with inverse astroglia numbers and few microglia. The number of neurons (including motoneurons) and astrocytes was stable after birth. Like in the spinal cord, motoneurons in organotypic spinal culture were <10% of ventral horn cells, with neurons <40%, and the rest made up by glia. The present report indicates a comparable degree of neuronal and glial maturation in brainstem and spinal motor nuclei, and that this condition is also observed in 3-week-old organotypic cultures. (c) 2011 Wiley Periodicals, Inc. Develop Neurobiol, 201
