Blood-brain barrier opening to large molecules does not imply blood-brain barrier opening to small ions.

Neuroimaging of exogenous tracer extravasation has become the technique of choice in preclinical and clinical studies of blood-brain barrier permeability. Such tracers have a larger molecular weight than small ions, neurotransmitters and many drugs. Therefore, it is assumed that tracer extravasation indicates both permeability to these and the cancelation of the electrical polarization across the barrier. Electrophysiological anomalies following intracarotideal administration of dehydrocholate, a bile salt causing extravasation of the albumin-binding tracer Evans blue, seemingly supported this. By contrast, electron microscopic studies suggested a different hierarchical pattern of blood-brain barrier dysfunction, a milder degree of impairment being characterized by increased function of the transcellular pathway and a severe degree by opening of the tight junctions. This would imply that the extravasation of macromolecules can occur before disruption of the electrical barrier. However, functional evidence for this has been lacking. Here, we further investigated the electrophysiological anomalies following intracarotideal application of dehydrocholate in rats and found that it caused focal cerebral ischemia by middle cerebral artery thrombosis, the electrophysiological recordings being characteristic of long-lasting spreading depolarization. These observations indicated that intracarotideal dehydrocholate is not a suitable model to study the isolated dysfunction of the blood-brain barrier. Second, we studied the topical application of dehydrocholate to the brain and the application of mannitol into the carotid artery. In both models, we found significant extravasation of Evans blue but no changes in either extracellular potassium or the CO(2)-dependent intracortical direct current deflection. The latter is assumed to depend on the proton gradient across the barrier in rats which we confirmed in additional experiments in vivo and in vitro. The stability of the extracellular potassium concentration and the CO(2)-dependent direct current deflection are two functional tests which indicate the integrity of the electrical barrier. Hence, our results provide functional evidence that the blood-brain barrier opening to large molecules does not necessarily imply the opening to small ions consistent with the hierarchy of damage in the previous electron microscopic studies.

The gamut of blood flow responses coupled to spreading depolarization in rat and human brain: from hyperemia to prolonged ischemia.

Cortical spreading depolarizations (SD) have been shown to occur frequently in patients with aneurysmal subarachnoid hemorrhage (SAH) and are associated with delayed ischemic brain damage. In animal models the link between SD and cell damage is the microvascular spasm coupled to the passage of SDs, resulting in spreading ischemia. Here we compared the hemodynamic changes induced by SD between human and rat cerebral cortex. Specifically, we addressed the question, whether the full spectrum of regional cerebral blood flow (rCBF) responses to SD is found in the human brain in a similar fashion to animal models. SDs were identified by slow potential changes in electrocorticographic recordings and the rCBF response profiles and magnitudes were analyzed. We found a large variability of rCBF changes concomitant to SDs in rat and in human recordings. The spectrum ranged from normal hyperemic responses to prolonged cortical spreading ischemia with intermediate forms characterized by biphasic (hypoemic-hyperemic) responses. The bandwidths of rCBF responses were comparable and the relative response magnitudes of hypo- and hyperperfusion phases did not differ significantly between rats and humans. The correspondence of the rCBF response spectrum to SD between human and animal brain underscores the importance of animal models to learn more about the mechanisms underlying the early and delayed pathological sequelae of SAH.

CGRP release and c-fos expression within trigeminal nucleus caudalis of the rat following glyceryltrinitrate infusion.

Neuropeptide release and the expression of c-fos like immunoreactivity (c-fos LI) within trigeminal nucleus caudalis neurons (TNC) are activation markers of the trigeminal nerve system. Glyceryltrinitrate (GTN) is believed to stimulate the trigeminal nerve system, thereby causing headache. We examined the effects of a 30 min NO-donor infusion on CGRP release in jugular vein blood and c-fos LI within TNC of the rat. GTN (2 and 50 microg/kg/min) or NONOate infusion (25 nmol/kg/min) did not cause any CGRP release during and shortly after infusion, whereas administration of capsaicin resulted in strongly increased CGRP levels. GTN infusion (2 microg/kg/min for 30 min) did not lead to enhanced c-fos LI after 2 h and 4 h, whereas capsaicin infusion caused a time- and dose-dependent expression of c-fos LI within laminae I and II of the TNC. Surprisingly, GTN attenuated capsaicin-induced c-fos expression by 64%. The nitric oxide synthase (NOS) inhibitor L-NAME (5 and 50 mg/kg) reduced capsaicin-induced c-fos LI dose dependently (reduction by 13% and 59%). We conclude that GTN may lead to headaches by mechanisms independent of CGRP release from trigeminal nerve fibres. GTN doses comparable to those used in humans did not activate or sensitize the trigeminal nerve system. Both GTN and L-NAME reduced capsaicin-induced c-fos LI. This is most likely due to a feedback inhibition of nitric oxide synthases, which indicates that the c-fos response to capsaicin within TNC is mediated by NO dependent mechanisms.

Differential patterns of expression of Eps15 and Eps15R during mouse embryogenesis.

Eps15 and Eps15R are related tyrosine kinase substrates, which have been implicated in endocytosis and synaptic vesicle recycling. Through the protein:protein interaction abilities of their EH domains, they establish a complex network of interactions with several proteins, including Numb, a protein necessary for neuronal cell fate specification. We analyzed the expression of Eps15 and Eps15R during murine development, at the time of active neurogenesis. The most striking difference was at the level of subcellular localization, with Eps15 present in the cytosol and on the plasma membrane, while Eps15R exhibited mainly a nuclear localization. Interesting topographical differences also emerged. In the 12.5 days post coitum neuroepithelium, Eps15 was expressed in the ventricular zone, which contains proliferating neuroblasts, whereas Eps15R was found only in postmitotic neurons. Conversely, both proteins were expressed in sensory and cranial ganglia. At later times, the expression of Eps15 and Eps15R was widely maintained in neuronal structures. In other tissues, Eps15 was first seen in the liver primordium and at low levels in choroid plexus, lung, kidney and intestine; later on the expression was maintained at high levels in epithelia. Nuclear staining of Eps15R was present in kidney, intestine, lung and liver, as well as in heart and pancreas.

Selective activation of NF-kappa B by nerve growth factor through the neurotrophin receptor p75.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) selectively bind to distinct members of the Trk family of tyrosine kinase receptors, but all three bind with similar affinities to the neurotrophin receptor p75 (p75NTR). The biological significance of neurotrophin binding to p75NTR in cells that also express Trk receptors has been difficult to ascertain. In the absence of TrkA, NGF binding to p75NGR activated the transcription factor nuclear factor kappa B (NF-kappa B) in rat Schwann cells. This activation was not observed in Schwann cells isolated from mice that lacked p75NTR. The effect was selective for NGF; NF-kappa B was not activated by BDNF or NT-3.

Selective binding and internalisation by truncated receptors restrict the availability of BDNF during development.

The tyrosine kinase receptor trkB is thought to mediate the biological actions of brain-derived neurotrophic factor. This receptor is expressed by a large variety of neurons during development. Truncated trkB molecules lacking the tyrosine kinase domain have also been described, but their functions remain elusive. In order to gain insight into their role, we studied the pattern of expression and properties of these truncated receptors in the chick embryo. mRNA coding for truncated trkB was detected already early during neurogenesis and in situ hybridisation experiments indicated that the expression was in non-neuronal cells, as previously observed in the brain of adult rodents. Ependymal and leptomeningeal cells expressing high levels of truncated trkB were found to completely surround the developing brain and the spinal cord throughout development. In the otic vesicle, mesenchymal cells expressing truncated trkB surround cells producing brain-derived neurotrophic factor, as well as neurons expressing trkB with its tyrosine kinase domain. Non-neuronal cells were found not to express trkB mRNA coding for the tyrosine kinase domain. Studies with radioiodinated brain-derived neurotrophic factor performed on frozen sections of the chick embryo revealed that non-neuronal cells expressing truncated trkB bind brain-derived neurotrophic factor with high affinity and selectivity. In addition, experiments with dissociated leptomeningeal cells revealed that binding is rapidly followed by selective internalisation of the ligand. These results suggest that truncated trkB molecules form an efficient and selective barrier preventing the diffusion of brain-derived neurotrophic factor and eliminating it by internalisation.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental regulation of full-length trkC in the rat sciatic nerve.

In order to gain insight into potential roles of neurotrophins in Schwann cell biology, the expression of neurotrophin receptors of the trk gene family was investigated in rat sciatic nerve development. This analysis revealed differential regulation of truncated and full-length receptors. TrkA was undetectable even when analysed with a sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) method. TrkB was present at the mRNA as well as protein level only in its truncated form. Surprisingly, multiple isoforms of trkC, including full-length forms, were detected in early postnatal nerve. Specific antibodies detected truncated and full-length trkC proteins in Western blotting, and RT-PCR revealed the presence of two full-length isoforms, one of them containing the 14 amino acid kinase insert. In situ hybridisation localized the expression of trkC to a subpopulation of Schwann cells. TrkC receptors are expressed already in nerves from day-16 embryos. In contrast to early postnatal stages, full-length trkC receptors are no longer expressed in adult nerves, which, however, maintain expression of truncated trkC transcripts. The presence of trkC kinases in peripheral nerve suggests a role for neurotrophin-3, the only known trkC ligand, in peripheral nerve development.