Miranda Mladinic

Kainate and metabolic perturbation mimicking spinal injury differentially contribute to early damage of locomotor networks in the in vitro neonatal rat spinal cord.

Acute 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.

Kainate-induced delayed onset of excitotoxicity with functional loss unrelated to the extent of neuronal damage in the in vitro spinal cord.

While excitotoxicity is a major contributor to the pathophysiology of acute spinal injury, its time course and the extent of cell damage in relation to locomotor network activity remain unclear. We used two in vitro models, that is, the rat isolated spinal cord and spinal organotypic cultures, to explore the basic characteristics of excitotoxicity caused by transient application of the glutamate analogue kainate followed by washout and analysis 24 h later. Electrophysiological records showed that fictive locomotion was slowed down by 10 microM kainate (with no histological loss) and fully abolished by 50 microM, while disinhibited bursting with unchanged periodicity persisted. Kainate concentrations (> or =50 microM) larger than those necessary to irreversible suppress fictive locomotion could still elicit dose-dependent motoneuron pool depolarization, and dose-dependent neuronal loss in the grey matter, especially evident in central and dorsal areas. Motoneuron numbers were largely decreased. A similar regional pattern was detected in organotypic slices, as extensive cell loss was dose related and affected motoneurons and premotoneurons: the number of dead neurons (already apparent 1 h after kainate) grew faster with the higher kainate concentration. The histological damage was accompanied by decreased MTT formazan production commensurate with the number of surviving cells. Our data suggest locomotor network function was very sensitive to excitotoxicity, even without observing extensive cell death. Excitotoxicity developed gradually leaving a time window in which neuroprotection might be attempted to preserve circuits still capable of expressing basic rhythmogenesis and reconfigure their function in terms of locomotor output.

O trauma raquimedular

A medula espinhal dos mamiferos adultos nao permite a regeneracao de axonios. Por razoes ainda desconhecidas, as fibras neurais falham em cruzar o sitio da lesao, como se nao houvesse crescimento, desde a primeira tentativa. Quais mecanismos poderiam explicar a perda da capacidade de regeneracao? As cicatrizes formadas pelas celulas da glia seriam uma consequencia da falha na regeneracao ou a causa? Diversas linhas de evidencia sugerem que a regeneracao da medula espinhal seria impedida no sistema nervoso central pela acao de fatores locais no sitio da lesao, e que o sistema nervoso central nao-lesado e um meio permissivo para o crescimento axonal, na direcao de alvos especificos. Uma vez que os axonios sao induzidos adequadamente a cruzar a lesao com o auxilio de implantes, farmacos ou celulas indiferenciadas, as fibras em regeneracao podem encontrar a via especifica e estabelecer conexoes corretas. O que ainda nao se sabe e que combinacao de moleculas induz/inibe o potencial de regeneracao do tecido e que mecanismos permitem aos neuronios formarem conexoes especificas com os alvos com os quais sao programados a fazer.

Postnatal developmental profile of neurons and glia in motor nuclei of the brainstem and spinal cord, and its comparison with organotypic slice cultures.

In 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 0–4 postnatal days, and its comparison with spinal organotypic cultures at 2–22 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.

Molecular Mechanisms Underlying Cell Death in Spinal Networks in Relation to Locomotor Activity After Acute Injury in vitro

Understanding the pathophysiological changes triggered by an acute spinal cord injury is a primary goal to prevent and treat chronic disability with a mechanism-based approach. After the primary phase of rapid cell death at the injury site, secondary damage occurs via autodestruction of unscathed tissue through complex cell-death mechanisms that comprise caspase-dependent and caspase-independent pathways. To devise novel neuroprotective strategies to restore locomotion, it is, therefore, necessary to focus on the death mechanisms of neurons and glia within spinal locomotor networks. To this end, the availability of in vitro preparations of the rodent spinal cord capable of expressing locomotor-like oscillatory patterns recorded electrophysiologically from motoneuron pools offers the novel opportunity to correlate locomotor network function with molecular and histological changes long after an acute experimental lesion. Distinct forms of damage to the in vitro spinal cord, namely excitotoxic stimulation or severe metabolic perturbation (with oxidative stress, hypoxia/aglycemia), can be applied with differential outcome in terms of cell types and functional loss. In either case, cell death is a delayed phenomenon developing over several hours. Neurons are more vulnerable to excitotoxicity and more resistant to metabolic perturbation, while the opposite holds true for glia. Neurons mainly die because of hyperactivation of poly(ADP-ribose) polymerase-1 (PARP-1) with subsequent DNA damage and mitochondrial energy collapse. Conversely, glial cells die predominantly by apoptosis. It is likely that early neuroprotection against acute spinal injury may require tailor-made drugs targeted to specific cell-death processes of certain cell types within the locomotor circuitry. Furthermore, comparison of network size and function before and after graded injury provides an estimate of the minimal network membership to express the locomotor program.

γ-Aminobutyric acidA ρ receptor subunits in the developing rat hippocampus

The RT‐PCR approach was used to estimate the expression of γ‐aminobutyric acid (GABA)A ρ receptor subunits in the hippocampus of neonatal and adult rats. All three ρ subunits were detected at postnatal day (P) 2, the ρ3 subunit being expressed at an extremely low level. The ρ1 and ρ2 products appeared to be developmentally regulated; they were found to be more pronounced in adulthood. In another set of experiments, to correlate gene expression with receptor function, GABAA ρ subunit mRNAs were detected with single‐cell RT‐PCR in CA3 pyramidal cells (from P3–P4 hippocampal slices), previously characterized with electrophysiological experiments for their bicuculline‐sensitive or ‐insensitive responses to GABA. In 6 of 19 cells (31%), pressure application of GABA evoked at –70 mV inward currents that persisted in the presence of 100 μM bicuculline (314 ± 129 pA). RT‐PCR performed in two of these neurons revealed the presence of ρ1 and ρ2 subunits, the latter being present with the α2 subunit. A ρ2 subunit was also found in 1 neuron (among 9) exhibiting a response to GABA, which was completely abolished by bicuculline. This might be due to the lack of putative accessory GABAA subunits that can coassemble with ρ2 to make functional receptors. Similar experiments from 10 P15 CA3 pyramidal cells failed to reveal any ρ1–3 transcripts. However, these neurons abundantly express α3 subunits. It is likely that in CA3 pyramidal cells of neonatal and adult hippocampus GABAA ρ subunits are present but at very low levels of expression.

Low expression of the ClC-2 chloride channel during postnatal development: a mechanism for the paradoxical depolarizing action of GABA and glycine in the hippocampus.

In early postnatal development, during the period of synapse formation, γ–aminobutyric (GABA) and glycine, the main inhibitory transmitters in the adult brain, paradoxically excite and depolarize neuronal membranes by an outward flux of chloride. The mechanisms of chloride homeostasis are not fully understood. It is known that in adult neurons intracellular chloride accumulation is prevented by a particular type of chloride channel, the ClC-2. This channel strongly rectifies in the inward direction at potentials negative to ECl thus ensuring chloride efflux. We have tested the hypothesis that in the developing hippocampus, a differential expression or regulation of ClC-2 channels may contribute to the depolarising action of GABA and glycine. We have cloned a truncated form of ClC-2 (ClC-2nh) from the neonatal hippocampus which lacks the 157 bp corresponding to exon 2. In situ hybridization experiments show that ClC-2nh is the predominant form of ClC-2 mRNA in the neonatal brain. ClC-2nh mRNA is unable to encode a full-length protein due to a frameshift, consequently it does not induce any currents upon injection into Xenopus oocytes. Low expression of the full–length ClC-2 channel, could alter chloride homeostasis, lead to accumulation of [Cl-]i and thereby contribute to the depolarizing action of GABA and glycine during early development.

Neuroprotection of locomotor networks after experimental injury to the neonatal rat spinal cord in vitro

Treatment to block the pathophysiological processes triggered by acute spinal injury remains unsatisfactory as the underlying mechanisms are incompletely understood. Using as a model the in vitro spinal cord of the neonatal rat, we investigated the feasibility of neuroprotection of lumbar locomotor networks by the glutamate antagonists 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) and aminophosphonovalerate (APV) against acute lesions induced by either a toxic solution (pathological medium (PM) to mimic the spinal injury hypoxic-dysmetabolic perturbation) or excitotoxicity with kainate. The study outcome was presence of fictive locomotion 24 h after the insult and its correlation with network histology. Inhibition of fictive locomotion by PM was contrasted by simultaneous and even delayed (1 h later) co-application of CNQX and APV with increased survival of ventral horn premotoneurons and lateral column white matter. Neither CNQX nor APV alone provided neuroprotection. Kainate-mediated excitotoxicity always led to loss of fictive locomotion and extensive neuronal damage. CNQX and APV co-applied with kainate protected one-third of preparations with improved motoneuron and dorsal horn neuronal counts, although they failed with delayed application. Our data suggest that locomotor network neuroprotection was possible when introduced very early during the pathological process of spinal injury, but also showed how the borderline between presence or loss of locomotor activity was a very narrow one that depended on the survival of a certain number of neurons or white matter elements. The present report provides a model not only for preclinical testing of novel neuroprotective agents, but also for estimating the minimal network membership compatible with functional locomotor output.

Dynamics of early locomotor network dysfunction following a focal lesion in an in vitro model of spinal injury.

It 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.

Mechanisms underlying cell death in ischemia-like damage to the rat spinal cord in vitro

New 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.