Critical illness polyneuropathy: clinical findings and cell culture assay of neurotoxicity assessed by a prospective study.

OBJECTIVE: First, to evaluate the role of typical intensive care-related conditions like sepsis, prolonged ventilation, drug effects and metabolic disorders in the pathogenesis of critical illness polyneuropathy (CIP); second, to investigate the possible significance of patient serum neurotoxicity assessed by an in vitro cytotoxicity assay with respect to CIP development. DESIGN: Prospective study. SETTING: Neurological intensive care unit. PATIENTS AND PARTICIPANTS: Twenty-eight patients who were on mechanical respiratory support for at least 4 days during a 21-month study period. RESULTS: Diagnosis of CIP was established by clinical and electrophysiological examination in 16 (57%) of 28 patients. Patients were investigated on days 4, 8 and 14 of mechanical ventilation. Two of 16 CIP patients had clinical signs of polyneuropathy at initial examination. Factors that correlated significantly with the development of CIP were: the multiple organ failure score on day 8 of ventilation, the total duration of respiratory support, the presence of weaning problems and the manifestation of complicating sepsis and/or lung failure. The in vitro toxicity assay showed serum neurotoxicity in 12 of 16 CIP patients. Electrophysiological investigations yielded false positive results of the toxicity assay in six patients (not developing CIP) and false negative results in four patients (developing clinical and electrophysiological signs of CIP). Statistical analysis did not reveal a significant correlation between the diagnosis of CIP and the finding serum neurotoxicity. CONCLUSION: The results support the hypothesis of a multi-factorial aetiopathogenesis of CIP. We observed serum neurotoxicity in the majority of CIP patients, indicating the possible involvement of a so far unknown, low-molecular-weight neurotoxic agent in CIP pathogenesis.

Cellular and subcellular localization of the 2B-subunit of the NMDA receptor in the adult rat telencephalon.

NMDA receptors (NR) are encoded by a family of genes including those of the NR1 and NR2A-D subunits. In situ hybridization has revealed that NR1, comprising eight splice variants, is ubiquitously expressed in the central nervous system (CNS) while the expression of NR2 isoforms is restricted to particular CNS regions. We report on the cellular and ultrastructural distribution of the NR2B polypeptide in rat telencephalon. In the telencephalon, the hippocampus represented the most intensively immunolabeled region. Here, predominantly the CA pyramidal neurons were heavily stained. Intense immunoreactivity (IR) was also detected in cortical neurons, in particular in pyramidal-like ones of layers II/III and V. On the ultrastructural level, the NR2B subunit was present not only in synaptic complexes where it usually was present in postsynaptic sites but in addition could be located at extrasynaptic sites. Furthermore, preliminary evidence indicates a presynaptic location of NR2B in some rare cases. NR2B antigen distribution is consistent with that of corresponding transcripts. Indeed, NR2B immunoreactivity coincides largely with that for NR1, indicating that both subunits are coexpressed in numerous cortical and hippocampal neurons.

The NMDA receptor subunit NR2B of neonatal rat brain: complex formation and enrichment in axonal growth cones.

The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors comprises a family of highly homologous subunits which assemble into oligomeric protein complexes. Alterations in subunit composition are developmentally regulated, leading to functionally distinct receptor populations. Here, the contribution of the subunit NR2B to NMDA receptor complex formation was analysed in neonatal rat brain, employing polyclonal antibodies raised against NR2B-specific synthetic peptides. By hydrodynamic size fractionation of the solubilized receptor protein and chemical cross-linking, NR2B antigen was found to be associated with several protein species of up to 690 kDa molecular weight. These observations show NR2B to be part of a multimeric receptor complex. Fractionation of cortex homogenates from E18 rat embryos on sucrose density gradients revealed NR2B polypeptide to be highly enriched in axonal growth cones. A similar distribution was found by fluorescence microscopy of immature hippocampal neurons, showing a preferential accumulation of NR2B antigen in axonal growth cones and varicosities. In mature cells, NR2B antigen displayed a punctated distribution pattern with redistribution to somato-dendritic spheres. The association of NR2B with axonal growth cones and processes of immature neurons suggests a role of NMDA receptors in the regulation of neurite outgrowth and migration.

Beta-bungarotoxin is a potent inducer of apoptosis in cultured rat neurons by receptor-mediated internalization.

The neurotoxic phospholipase A(2), beta-bungarotoxin (beta-BuTx), is a component of the snake venom from the Taiwanese banded krait Bungarus multicinctus. beta-BuTx affects presynaptic nerve terminal function of the neuromuscular junction and induces widespread neuronal cell death throughout the mammalian and avian CNS. To analyse the initial events of beta-BuTx-mediated cell death, the toxin was applied to cultured rat hippocampal neurons where it induced neuronal cell death in a concentration-dependent manner (EC(50) approximately equal to 5 x 10(-13) M) within 24 h. Fluorescence labelled beta-BuTx was completely incorporated by neurons within < 10 min. Binding and uptake of beta-BuTx, as well as induction of cell death, were efficiently antagonized by preincubation with dendrotoxin I, a blocker of voltage-gated potassium channels devoid of phospholipase activity. Binding of beta-BuTx was selective for neurofilament-positive cells. As evident from intense annexin-V and TUNEL stainings, application of beta-BuTx induced apoptotic cell death exclusively in neurons, leaving astrocytes unaffected. No evidence was obtained for any contribution of either caspases or calpains to beta-BuTx-induced apoptosis, consistent with the inability of the inhibitors Z-Asp-DCB and calpeptin, respectively, to protect neurons from beta-BuTx-induced cell death. These observations indicate that induction of cell death by beta-BuTx comprises several successive phases: (i) binding to neuronal potassium channels is the initial event, followed by (ii) internalization and (iii) induction of apoptotic cell death via a caspase-independent pathway.

Transient expression of NMDA receptor subunit NR2B in the developing rat heart.

NMDA receptors represent a subtype of the ionotropic glutamate receptor family, comprising three classes of subunits (NR1, NR2A-D, NR3), which exhibit distinct patterns of regional and developmental expression in the CNS. Recently, some NMDA receptor subunits have also been described in adult extraneuronal tissues and keratinocytes. However, their developmental expression patterns are currently unknown. With use of RT-PCR and western blot analysis, the expression of NMDA receptor subunit NR2B was investigated in the developing rat heart. NR2B mRNA and protein were detected in heart tissue of rats from embryonic day 14 until postnatal day 21 but disappeared 10 weeks after birth. In contrast, no NMDA receptor subunit NR1, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunit GluR2, or anchoring postsynaptic density protein-95 could be detected in rat heart at any developmental stage. Confocal microscopy of cultured cardiac myocytes (CMs) from neonatal rats revealed distinct NR2B staining mainly of intracellular structures. However, no functional NMDA receptor could be detected on CMs by whole-cell recordings. In conclusion, high concentrations of NR2B protein can be detected in early rat heart development, but its function still remains elusive.