A simple novel approach for detecting blood-brain barrier permeability using GPCR internalization.

AIMS: Impairment of blood-brain barrier (BBB) is involved in numerous neurological diseases from developmental to aging stages. Reliable imaging of increased BBB permeability is therefore crucial for basic research and preclinical studies. Today, the analysis of extravasation of exogenous dyes is the principal method to study BBB leakage. However, these procedures are challenging to apply in pups and embryos and may appear difficult to interpret. Here we introduce a novel approach based on agonist-induced internalization of a neuronal G protein-coupled receptor widely distributed in the mammalian brain, the somatostatin receptor type 2 (SST2). METHODS: The clinically approved SST2 agonist octreotide (1 kDa), when injected intraperitoneally does not cross an intact BBB. At sites of BBB permeability, however, OCT extravasates and induces SST2 internalization from the neuronal membrane into perinuclear compartments. This allows an unambiguous localization of increased BBB permeability by classical immunohistochemical procedures using specific antibodies against the receptor. RESULTS: We first validated our approach in sensory circumventricular organs which display permissive vascular permeability. Through SST2 internalization, we next monitored BBB opening induced by magnetic resonance imaging-guided focused ultrasound in murine cerebral cortex. Finally, we proved that after intraperitoneal agonist injection in pregnant mice, SST2 receptor internalization permits analysis of BBB integrity in embryos during brain development. CONCLUSIONS: This approach provides an alternative and simple manner to assess BBB dysfunction and development in different physiological and pathological conditions.

Early Recruitment of Cerebral Microcirculation by Neuronal Nitric Oxide Synthase Inhibition in a Juvenile Ischemic Rat Model.

BACKGROUND: The development of collateral circulation is proposed as an inherent compensatory mechanism to restore impaired blood perfusion after ischemia, at least in the penumbra. We have studied the dynamic macro- and microcirculation after ischemia-reperfusion in the juvenile rat brain and evaluated the impact of neuronal nitric oxide synthase (nNOS) inhibition on the collateral flow. METHODS: Fourteen-day-old (P14) rats were subjected to ischemia-reperfusion and treated with either PBS or 7-nitroindazole (7-NI, an nNOS inhibitor, 25 mg/kg). Arterial blood flow (BF) was measured using 2D-color-coded pulsed ultrasound imaging. Laser speckle contrast (LSC) imaging and sidestream dark-field videomicroscopy were used to measure cortical and microvascular BF, respectively. RESULTS: In basal conditions, 7-NI reduced BF in the internal carotids (by ∼ 25%) and cortical (by ∼ 30%) BF, as compared to PBS. During ischemia, the increased mean BF velocity in the basilar trunk after both PBS and 7-NI demonstrated the establishment of collateral support and patency. Upon re-flow, BF immediately recovered to basal values in the internal carotid arteries under both conditions. The 7-NI group showed increased collateral flow in the penumbral tissue during early re-flow compared to PBS, as shown with both LSC imaging and side-stream dark-field videomicroscopy. The proportion of perfused capillaries was significantly increased under 7-NI as compared to PBS when given before ischemia (67.0 ± 3.9 vs. 46.8 ± 8.8, p < 0.01). Perfused capillaries (63.1 ± 17.7 vs. 81.1 ± 20.7, p < 0.001) and the BF index (2.4 ± 0.6 vs. 1.3 ± 0.1, p < 0.001) significantly increased under 7-NI given at the re-flow onset. CONCLUSIONS: Collateral support in the penumbra is initiated during ischemia, and may be increased during early re-flow by neuronal NOS inhibition (given in pre- and post-treatment), which may preserve brain tissue in juvenile rats.

Stroke induces histamine accumulation and mast cell degranulation in the neonatal rat brain.

Inflammatory processes are a major cause of hypoxic-ischemic brain damage. The present study focuses on both the cerebral histamine system and mast cells in a model of transient focal ischemia induced by permanent left middle cerebral artery, and homolateral transient common carotid artery occlusion (50 minutes) in the P7 newborn rat. Immunohistochemical analysis revealed that ischemia induces histamine (HA) accumulation in the core of the infarct 6-12 h post-ischemia, and in the penumbra at 24-48 h, although in situ hybridization failed to detect any histidine decarboxylase gene transcripts in these regions. Immunohistochemical co-localization of HA with the MAP2 marker revealed that HA accumulates in neuronal cells before they degenerate, and is accompanied by a very significant increase in the number of mast cells at 12 h and 48 h of reperfusion. In mast cells, histamine immunoreactivity is detected at 2, 6 and 12 h after ischemia, whereas it disappears at 24 h, when a concomitant degranulation of mast cells is observed. Taken together, these data suggest that the recruitment of cerebral mast cells releasing histamine may contribute to ischemia-induced neuronal death in the immature brain.

Apoptotic features of selective neuronal death in ischemia, epilepsy and gp 120 toxicity.

The occurrence of physiological cell death has been known for decades, but interest in the subject was renewed in 1972 when Kerr, Wyllie and Currie described in detail the ultrastructural changes characteristic of dying cells and coined the term apoptosis to describe the process. Cells display a wide variety of morphological changes when dying during development or following a toxic insult. A binary classification scheme suggests that physiologically appropriate death is due to apoptosis and that pathological mechanisms involve necrosis. However, recent studies indicate a potential involvement of apoptotic cell death in ischemia, status epilepticus and HIV-1 infection.

Early endonuclease activation following reversible focal ischemia in the rat brain.

The structural changes that occur in chromatin DNA after ischemic brain injury are poorly understood. The presence of oligonucleosome fragments that are recognized as the characteristic DNA ladder has been demonstrated in global and focal ischemia, associated or not with random DNA fragmentation. Using pulsed-field gel electrophoresis, which improves DNA separation, we have now detected initial stages of DNA fragmentation that occur already 6 h after reversible focal cerebral ischemia in rats. This result confirms that internucleosomal DNA fragmentation precedes random DNA fragmentation in vulnerable striatal and cortical neurons following reversible focal cerebral ischemia.

Apoptosis and necrosis after reversible focal ischemia: an in situ DNA fragmentation analysis.

Apoptosis is one of the two forms of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytological alternations including membrane blebbing and nuclear and cytoplasmic condensation. Early activation of an endonuclease has been previously demonstrated after a transient focal ischemia in the rat brain Charriaut-Marlangue C, Margaill I, Plotkine M, Ben-Ari Y (1995) Early endonuclease activation following reversible focal ischemia. J Cereb Blood Flow Metab 15:385-388). We now show that a significant number of striatal and cortical neurons, exhibited chromatin condensation, nucleus segmentation, and apoptotic bodies increasing with recirculation time, as demonstrated by in situ labeling of DNA breaks in cryostat sections. Apoptotic nuclei were also detected in the horizontal limb diagonal band, accumbens nucleus and islands of Calleja. Several necrotic neurons, in which random DNA fragmentation occurs, were also shown at 6 h recirculation, in the ischemic core. Further investigation with hematoxylin/eosin staining revealed that apoptotic nuclei were present in cells with a large and swelled cytoplasm and in cells with an apparently well-preserved cytoplasm. These two types of cell death were reminiscent of those described in developmental cell death. Our data suggested that apoptosis may contribute to the expansion of the ischemic lesion.

Increase in Specific Proteins and mRNAs Following Transient Anoxia - Aglycaemia in Rat CA1 Hippocampal Slices.

Incorporation of [35S]methionine into proteins and two-dimensional gel autoradiograms was used to characterize early post-anoxia - aglycaemia protein synthesis in the CA1 area of rat hippocampal slices maintained in vitro. We have compared the effects of 3 - 4 min and 5 - 10 min insults, since the former but not the latter produces a reversible block of synaptic transmission (see companion paper). An insult of between 3 min 30 s and 4 min induces a transient increase in the labelled proteins during the first hour of reoxygenation, as compared to control. The increase in protein synthesis is conspicuous for several proteins, including actin, alpha-tubulin and heat-shock proteins (hsp70c and hsp90), as determined by immunoblotting. In the case of alpha-tubulin, we show with in situ hybridization and polymerase chain reaction procedures that the increase in protein synthesis is associated with a marked increase in the expression of the corresponding messenger RNAs. The results demonstrate that, in addition to regulatory proteins such as hsps, the synthesis of several polypeptides, including those associated with the cytoskeleton, is altered in anoxic damage.

Long-term potentiation in the rat hippocampus induced by the mast cell degranulating peptide: analysis of the release of endogenous excitatory amino acids and proteins.

Using a push-pull device, we have analysed, in vivo, the release of endogenous excitatory amino acids and proteins induced by the mast cell degranulating peptide in the CA1 region of the hippocampus. Local application of the mast cell degranulating peptide (20 microM) for 5 or 10 min produced a long-term potentiation of the slope of the field excitatory postsynaptic potential (70 +/- 40%, 3 h after the drug application). This long-term potentiation was associated with (i) a transient increase (10 min) in the release of endogenous glutamate and aspartate and (ii) a late transient enhanced release of proteins and newly secreted proteins. In cases in which the mast cell degranulating peptide induced recurrent interictal activity, there was a sustained enhanced release of glutamate. These observations suggest that mast cell degranulating peptide induced long-term potentiation is not associated with a sustained enhanced release of excitatory amino acids.

Distribution of Aquaporin 9 in the adult rat brain: preferential expression in catecholaminergic neurons and in glial cells.

Aquaporin 9 (AQP9) is a recently cloned water channel that is permeable to monocarboxylate, glycerol and urea. In rat, AQP9 has been found in testis and liver as well as in brain where its expression has been initially shown in glial cells in forebrain. However, the expression of AQP9 has not been investigated in the brainstem. The purpose of this study is to describe the distribution of AQP9-immunoreactive cells throughout the adult rat brain using reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry. We performed immunolabeling on brain from animals perfused with fixative and we show that AQP9 is expressed (i) in astrocytes in the glia limitans, in the white matter and in glial cells of the cerebellum, (ii) in the endothelial cells of pial vessels, and (iii) in specific groups of neurons. The neuronal AQP9 expression was almost exclusively observed in catecholaminergic cells including the adrenergic, noradrenergic and dopaminergic groups, but not in other monoaminergic neurons such as serotonergic or histaminergic cells. A slight labeling was also observed in non-catecholaminergic neurons localized in the paraventricular nucleus of the hypothalamus. These results indicate that AQP9 has a unique brain distribution with a preferential localization in catecholaminergic nuclei known to be involved in many cerebral functions. While the presence of AQP9 in glia limitans and in endothelial cells of the pial vessels could be related to water transport through the blood-brain barrier, its expression in neuronal cells, not directly involved in the osmoregulation, suggests that brain AQP9 could also be used as a metabolite channel since lactate and glycerol can be energy substrates for neurons.

Kainate-induced apoptotic cell death in hippocampal neurons.

We have examined the role apoptosis plays in epileptic brain damage using intra-amygdaloid injection of kainate. With the silver staining technique of Gallyas, argyrophylic (dying) neurons were observed, a few hours after the injection, in the amygdala and in the vulnerable pyramidal neurons of the hippocampal CA3 region. In both areas, cell death has apoptotic features, including: (i) nuclear chromatin condensation and marginalization with light and electron microscopy; (ii) DNA fragmentation with a typical ladder pattern on agarose gel electrophoresis; (iii) positive nuclear labelling with a selective in situ DNA fragmentation staining method. Combined in situ DNA labelling and silver staining showed that the DNA fragmentation occurred in dying neurons. CA1 or granule cells which do not degenerate following intra-amygdaloid injection of kainate were not stained with the in situ DNA labelling or the argyrophylic technique. Administration of diazepam blocked the kainate-induced seizures and prevented DNA fragmentation in CA3 but not in the amygdala. Therefore, apoptosis contributes to the local and distant damage induced by kainate.

Loss of NADPH-diaphorase containing neurones after reversible focal ischaemia in rats delayed by L-NAME.

In the present study, NADPH-diaphorase histochemistry was used to assess the temporal evolution of the number of nitric oxide (NO)-synthase containing neurones after reversible focal cerebral ischaemia in rats. The number of NADPH-diaphorase containing neurones was reduced by 50% and 90% respectively 6 and 24 h after ischaemia. L-NAME, a NO-synthase inhibitor, prevented the loss of NADPH-diaphorase containing neurones observed 6 h after ischemia but not 24 h after ischaemia, suggesting that in the early phase, nitric oxide is involved in this phenomenon.

A cautionary note on the use of the TUNEL stain to determine apoptosis.

Detection of DNA fragments in situ using the terminal deoxyribonucleotidyl transferse (TdT)-mediated biotin-16-dUTP nick-end labelling (TUNEL assay) is now commonly used to investigate apoptosis. Previous reports suggested that physiologically appropriate death is due to apoptosis and that pathological mechanisms involve necrosis. Strong evidence of apoptosis, following ischaemia and epilepsy, has been recently provided by combining genomic DNA gel electrophoresis, light and electron microscopy and in situ, DNA-break labelling. However, only an observation in light microscopy with high magnification permits the detection of chromatin condensation and apoptotic bodies. We report that a positive TUNEL assay reaction should not be considered as a specific marker of apoptosis but can also indicate necrotic cell death.

Inhibition of protein synthesis by the NMDA channel blocker MK-801.

MK-801 is a selective non-competitive N-methyl-D-aspartate (NMDA) channel blocker which has been extensively used in order to determine the properties of NMDA receptors and their role in epilepsy and ischaemic cell death. We now report that MK-801 (10 microM) inhibits [35S]methionine incorporation into polypeptides by 45 +/- 7.5% in adult rat hippocampal slices and by 35 +/- 10% in cultured glial cells. This effect is NMDA-receptor independent since (i) only high doses of MK-801 induced such an effect and (ii) APV (50 microM), a competitive NMDA receptor antagonist, or Mg2+ (10 mM), a NMDA channel blocker, did not reduce protein synthesis.

Apoptosis associated DNA fragmentation in epileptic brain damage.

We have examined the epileptic brain damage generated by intra-amygdaloid administration of kainic acid (KA). In the vulnerable CA3 region of the rat hippocampus, neurones developed apoptotic properties since, (i) their nuclei were positively stained with a selective in situ DNA fragmentation staining method; (ii) DNA cleavage into internucleosome-sized fragments was observed on agarose gels. Our results suggest that epileptic seizures generated in the amygdala by KA trigger apoptotic cell damage in pyramidal CA3 neurones.

The HIV-1 envelope protein gp120 induces neuronal apoptosis in hippocampal slices.

The HIV-1 envelope protein gp120 produces neuronal cell damage in primary cultures of a variety of cell types including hippocampal and retinal ganglion cell neurons. The properties of primary cell cultures are, however, often markedly different from those of cells living in their normal environment. We now report that gp120 induces widespread chromatin condensation and lesions in pyramidal granular neurones and in interneurones of rat hippocampal organotypic slice cultures. This damage is clearly of an apoptotic (programmed cell death) type. The use of an in vitro organized structure will enable the molecular and cellular mechanism of action of gp120 to be examined in conditions which are particularly suitable and relevant to the in vivo situation.

Increased synthesis of specific proteins during glutamate-induced neuronal death in cerebellar culture.

We have previously shown that glutamate-induced neurotoxicity is mediated by a sodium-chloride component and a calcium component in our cerebellar granule cell culture. In order to further characterize these two different components, the time course of neuronal death induced by glutamate (100 microM) in basal solution and in low sodium-chloride solution was studied by morphological and biochemical criteria. As shown by phase-contrast microscopy, cerebellar granule cells exhibited clear neuronal degeneration within 4 h after exposure to this excitotoxin. These morphological changes correlated [35S]methionine incorporation into proteins which rapidly declined during the first hour of treatment. Qualitative change in [35S]methionine incorporation into proteins was further investigated by two-dimensional gel electrophoresis performed after glutamate exposure in basal solution and in low sodium-chloride solution. Most of the proteins showed a decreased labelling after glutamate exposure as expected, but some polypeptides showed an increased labelling or appeared to be newly synthesized. Furthermore, a different pattern of protein synthesis was observed when glutamate exposure was performed in basal solution or in low sodium-chloride solution. The identification of these polypeptides and their implication in the neuronal death are discussed.

Anisomycin and cycloheximide protect cerebellar neurons in culture from anoxia.

Protein synthesis inhibitors have recently been shown to protect from ischemia-induced neuronal death in the rat hippocampus in vivo. In an attempt to further investigate the mechanism of neuronal death resulting from anoxia, cerebellar neurons grown in culture were exposed to an anoxic atmosphere in the presence of protein synthesis inhibitors. Anisomycin and cycloheximide (100 micrograms/ml) offered, respectively, a 97 +/- 4% and 26 +/- 13% protection against anoxia-induced neuronal death.

Glutamate-induced neuronal death in cerebellar culture is mediated by two distinct components: a sodium-chloride component and a calcium component.

The relative contribution of sodium, chloride and calcium ions in the neuronal death induced by glutamate is controversial. We have therefore reassessed the effects of extracellular ion substitution on glutamate-induced neuronal death in cerebellar granule cell culture. Sodium or chloride substitution by impermeant ions prevented the initial swelling observed after glutamate exposure (100 microM, 15 min) in balanced salt solution but did not prevent the progressive degeneration of cerebellar neurons over the next few hours. In low calcium medium, glutamate exposure also led to degeneration of granule neurons. In contrast, sodium or chloride substitution and calcium omission prevented both the initial swelling and the delayed neuronal death after glutamate exposure. These morphological observations were confirmed both by measurement of the intracellular water space with [3H]methylglucose and by quantification of cell viability by 3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyl tetrazolium bromide (MTT) staining. We conclude that glutamate-induced neuronal death is mediated by two distinct components: a calcium-independent sodium-chloride dependent component and a calcium-dependent component. Each one of these components leads to the death of cerebellar neurons after glutamate exposure.

Transient increased density of NMDA binding sites in the developing rat hippocampus.

Using quantitative autoradiography and membrane preparations, the density of specific glutamate and N-methyl-D-aspartic acid (NMDA) binding sites have been determined in the developing rat hippocampus. We found an abrupt reduction in the density of NMDA binding sites after P8 (postnatal day) without change in affinity. The transient expression of NMDA receptors during maturation suggests that they may play a particularly important role in synaptogenesis.

Down-regulation of striatal enkephalinergic (PPA) messenger RNA without prior apoptotic features following reversible focal ischemia in rat.

In order to determine whether striatal enkephalinergic neurons were affected by reversible focal ischemia, we have investigated the expression of the preproenkephalin (PPA) messenger by in situ hybridization (ISH) combined with TUNEL staining to display apoptosis in the same rat brain sections. Our data demonstrated a massive reduction of the number of PPA-mRNA containing neurons concomitant with the emergence of apoptotic cells. However, double-labeled neurons (ISH- and TUNEL-positive cells) were not detected, suggesting that either disruption of mRNA precedes DNA fragmentation or ischemia leads to a long lasting reduction of mRNA(s) without damage.