Purines induce directed migration and rapid homing of microglia to injured pyramidal neurons in developing hippocampus

Traumatic CNS injury activates and mobilizes resident parenchymal microglia (MG), which rapidly accumulate near injured neurons where they transform into phagocytes. The mechanisms underlying this rapid 'homing' in situ are unknown. Using time-lapse confocal imaging in acutely excised neonatal hippocampal slices, we show that rapid accumulation of MG near somata of injured pyramidal neurons in the stratum pyramidale (SP) results from directed migration from tissue regions immediately adjacent to (<200 microm from) the SP. Time-lapse sequences also reveal a 'spreading activation wave' wherein MG situated progressively farther from the SP begin to migrate later and exhibit less directional migration toward the SP. Because purines have been implicated in MG activation and chemotaxis, we tested whether ATP/ADP released from injured pyramidal neurons might account for these patterns of MG behavior. Indeed, application of apyrase, which degrades extracellular ATP/ADP, inhibits MG motility and homing to injured neurons in the SP. Moreover, bath application of exogenous ATP/ADP disrupts MG homing by inducing directional migration toward the slice exterior and away from injured neurons. These results indicate that extracellular ATP/ADP is both necessary and sufficient to induce directional migration and rapid homing of neonatal MG to injured neurons in situ. Rapid, ATP/ADP-dependent MG homing may promote clearance of dead and dying cells and help limit secondary damage during the critical first few hours after neuronal injury.     

Differentiation-dependent sensitivity to cell death induced in the developing retina by inhibitors of the ubiquitin-proteasome proteolytic pathway

The effects of inhibitors of proteasome function were studied in the retina of developing rats. Explants from the retina of neonatal rats at postnatal day (P) 3 or P6 were incubated with various combinations of the proteasome inhibitor carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132), the protein synthesis inhibitor anisomycin, or the adenylyl cyclase activator forskolin. MG132 induced cell death in a subset of cells within the neuroblastic (proliferative) layer of the retinal tissue. The cells sensitive to degeneration induced by either MG132 or anisomycin, were birthdated by bromodeoxyuridine injections. This showed that the MG132-sensitive population includes both proliferating cells most likely in their last round of cell division, and postmitotic undifferentiated cells, at a slightly earlier stage than the population, sensitive to anisomycin-induced cell death. The results show that sensitivity to cell death induced by proteasome inhibitors defines a window of development in the transition from the cell cycle to the differentiated state in retinal cells.     

Induction of rapid,activity-dependent neuronal-glial remodelling in the adult rat hypothalamus in vitro

The hypothalamic oxytocinergic system offers a remarkable model of morphological plasticity in the adult because its neurons and astrocytes undergo mutual remodelling in relation to differing physiological conditions. Among various factors involved in such plasticity, oxytocin (OT) itself appears of primary importance as its central administration resulted in morphological changes similar to those brought on by physiological stimuli. In the present study, we applied OT on acute hypothalamic slices from adult rats that included the supraoptic nucleus. Using ultrastructural morphometric analyses, we found that it induced a significant reduction of astrocytic coverage of OT neurons, leaving their surfaces directly juxtaposed, to an extent similar to that detected in vivo under conditions like lactation. These neuronal-glial changes were rapid and reversible, occurring within a few hours, and specifically mediated via OT receptors. They were potentiated by oestrogen and depended on calcium mobilization and de novo protein synthesis. Moreover, they depended on concurrent neuronal activation brought on by hyperosmotic stimulation or blockade of inhibitory GABAergic neurotransmission; they were inhibited by blockade of glutamatergic receptors. Taken together, our observations show that intrahypothalamic release of OT affects not only neuronal activation of the OT system but its morphological plasticity as well. Moreover, the activity dependence of the OT-induced changes strongly suggests that astrocytes can sense the level of activity of adjacent neurons and/or afferent input and this can subsequently act as a signal to bring on the neuronal and glial conformational changes.     

Activity-dependent regulation of a ribosomal RNA epitope in the chick cochlear nucleus

Elimination of auditory nerve activity results in rapid metabolic changes, cell atrophy, and cell death in nucleus magnocellularis (NM), the cochlear nucleus of the chick. The transneuronal signals involved in the activity-dependent regulation of NM neurons are not well understood. One of the most rapid transneuronal effects is alteration in protein synthesis by NM neurons. Previous studies using an in vitro preparation of the brain stem auditory system suggested that up-regulation of protein synthesis in NM neurons requires the action of some trophic substance released by active auditory nerve fibers. Here, similar results were obtained when measuring changes in immunoreactivity using a monoclonal antibody (Y10B) that recognizes ribosomal RNA. This immunolabeling assay has advantages over the global protein synthesis assay in that it is not sensitive to possible changes in specific activity of the precursor pool or possible differences in the uptake of the labeled amino acids. Unilateral stimulation of the auditory nerve for 1 h resulted in greater immunolabeling of NM neurons on the stimulated side of the slice. This is consistent with previous in vivo results after unilateral deafferentation. Blockade of synaptic transmission by maintaining the slice in a low-Ca²⁺/high Mg²⁺ medium prevented the stimulation-induced difference in immunolabeling. Electrical stimulation of the postsynaptic NM neurons alone (antidromic stimulation, via electrical stimulation of NM neuron axons) did not result in greater immunolabeling. Rather, antidromically stimulated neurons tended to show lighter labeling. Thus, the transneuronal regulation of ribosomes in NM neurons appears to require some substance released from the active auditory nerve. Further, blockade of protein synthesis using cycloheximide did not prevent the activity-dependent difference in immunolabeling. Thus, the activity-dependent regulation of ribosomes does not appear to require new protein synthesis during the period of differential activity.     

Neuroinflammatory responses after experimental diffuse traumatic brain injury

Little is known about microglial activation and macrophage localization after diffuse brain injury (DBI). DBI-mediated perisomatic traumatic axonal injury (TAI) was recently identified within the neocortex, hippocampus, and thalamus, providing an opportunity to characterize immune cell responses within diffusely injured brain loci uncomplicated by contusion. By using moderate midline/central fluid percussion injury, microglial/macrophage responses were examined with antibodies targeting immune cell phenotypes and amyloid precursor protein, a marker of TAI. Parallel assessments of blood-brain barrier alterations were also performed. Within 6 to 48 hours postinjury, microglial activation within injured loci was observed, whereas microglia within non-TAI-containing regions maintained a resting phenotype. Microglial activation shared a spatiotemporal relationship with TAI though no clear interactions were observed. By 7 to 28 days postinjury, activated microglia contained myelin debris, yet revealed limited aggregation. Immunophenotypic macrophages were also localized to injured loci. Select macrophages approximated somatic membranes of perisomatically axotomized neurons with evidence of bouton disruption. No causality was established between blood-brain barrier alterations and these inflammatory responses. These findings indicate rapid, yet initially nonspecific, and persistent microglial/macrophage responses to DBI. DBI-mediated inflammatory responses suggest further expansion of traumatic brain injury histopathologic evaluations to identify neuroinflammation indicative of diffuse pathology.     

Dynamics of microglial activation: a confocal time-lapse analysis in hippocampal slices

The dynamics of microglial cell activation was studied in freshly prepared rat brain tissue slices. Microglia became activated in the tissue slices, as evidenced by their conversion from a ramified to amoeboid form within several hours in vitro. To define better the cytoarchitectural dynamics underlying microglial activation, we performed direct three-dimensional time-lapse confocal imaging of microglial cells in live brain slices. Microglia in tissue slices were stained with a fluorescent lectin conjugate, FITC-IB(4), and stacks of confocal optical sections through the tissue were collected repeatedly at intervals of 2-5 min for several hours at a time. Morphometric analysis of cells from time-lapse sequences revealed that ramified microglia progress to amoeboid macrophages through a stereotypical sequence of steps. First, in the withdrawal stage, the existing ramified branches of activating microglia do not actively extend or engulf other cells, but instead retract back (mean rate, 0.5-1.5 microm/min) and are completely resorbed into the cell body. Second, in the motility stage, a new set of dynamic protrusions, which can exhibit cycles of rapid extension and retraction (both up to 4 microm/min), abruptly emerges. Sometimes new processes begin to emerge even before the old branches are completely withdrawn. Third, in the locomotory stage, microglia begin translocating within the tissue (up to 118 microm/h) only after the new protrusions emerge. We conclude that the rapid conversion of resting ramified microglia to active amoeboid macrophages is accomplished not by converting quiescent branches to dynamic ones, but rather by replacing existing branches with an entirely new set of highly motile protrusions. This suggests that the ramified branches of resting microglia are normally incapable of rapid morphological dynamics necessary for activated microglial function. More generally, our time-lapse observations identify changes in the dynamic behavior of activating microglia and thereby help define distinct temporal and functional stages of activation for further investigation.     

Acute activation of mitogen-activated protein kinases following traumatic brain injury in the rat: implications for posttraumatic cell death

The regional activation (via phosphorylation) of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways was examined using immunoblotting and immunohistochemistry following experimental brain injury. Anesthetized rats were subjected to lateral fluid-percussion brain injury of moderate severity (2.4-2.6 atm) and euthanized at 2, 6, 24, and 72 h after injury; sham-injured animals were surgically prepared but were not injured. Immunohistochemical evidence of activation of JNK and ERK1/2 pathways was observed predominantly in regions that exhibit neural cell apoptosis and axonal damage following brain trauma. Activation of the ERK1/2 pathway was observed as early as 2 h and up to 72 h postinjury in nonneuronal cells in all layers of the cortex at the site of maximal injury, in the white matter below the site of maximal cortical damage and in the thalamus. In contrast, activation of JNK signaling was observed only at 24 and 72 h postinjury in a few neurons at the core of the cortical injury site. However, robust JNK activation was observed between 2 and 72 h postinjury in both axons and nonneuronal cells in the white matter below the site of maximal cortical damage and in the thalamus. Activation of ERK1/2, but not JNK, was observed in cells in the dentate hilus in the hippocampus in both hemispheres between 2 and 24 h postinjury. Immunoblotting analyses of extracts from various brain regions did not reveal significant alterations in intensities of either total or phosphorylated proteins underscoring the focal nature of the immunohistochemical observations. However, these results suggest that activation of MAP kinase signaling pathways may be associated with posttraumatic cell damage and are indicative of the heterogeneous nature of the mechanisms underlying regional cell death following TBI.     

Effects of oxygen-glucose deprivation on microglial mobility and viability in developing mouse hippocampal tissues

As brain‐resident immune cells, microglia (MG) survey the brain parenchyma to maintain homeostasis during development and following injury. Research in perinatal stroke, a leading cause of lifelong disability, has implicated MG as targets for therapeutic intervention during stroke. Although MG responses are complex, work in developing rodents suggests that MG limit brain damage after stroke. However, little is known about how energy‐limiting conditions affect MG survival and mobility (motility and migration) in developing brain tissues. Here, we used confocal time‐lapse imaging to monitor MG viability and mobility during hypoxia or oxygen‐glucose deprivation (OGD) in hippocampal tissue slices derived from neonatal GFP‐reporter mice (CX3CR1^GFP/+). We found that MG remain viable for at least 6 h of hypoxia but begin to die after 2 h of OGD, while both hypoxia and OGD reduce MG motility. Unexpectedly, some MG retain or recover motility during OGD and can engulf dead cells. Additionally, MG from younger neonates (P2–P3) are more resistant to OGD than those from older ones (P6–P7), indicating increasing vulnerability with developmental age. Finally, transient (2 h) OGD also increases MG death, and although motility is rapidly restored after transient OGD, it remains below control levels for many hours. Together, these results show that MG in neonatal mouse brain tissues are vulnerable to both transient and sustained OGD, and many MG die within hours after onset of OGD. Preventing MG death may, therefore, provide a strategy for promoting tissue restoration after stroke. © 2012 Wiley Periodicals, Inc.     

Experimental fluid percussion brain injury: vascular disruption and neuronal and glial alterations

Because of the potential relationship between vascular disturbances and secondary tissue damage, we identified areas of brain which exhibited hemorrhage and leakage of protein during the acute stage after experimental brain injury and subsequently studied the development of pathologic changes, including cavity formation, neuronal necrosis, and gliosis within these regions. The development of pathologic changes was evaluated at 1, 6, and 24 h and 1, 2, and 4 weeks after lateral, fluid percussion (FP) brain injury of moderate severity in the rat. Vascular disruption in the acute stages, as evidenced by hemorrhage and leakage of Evans blue albumin, was most prominent 6 h postinjury and was maximal in the parieto-occipital cortex. From 1 to 24 h after injury, regions of the injured hemisphere, including the cortex and hippocampus, exhibited abnormal neurons which stained with acid fuchsin and Alizarin red, histochemical markers for injured neurons and calcium, respectively. These same regions suffered significant neuronal cell loss from 1 to 4 weeks after injury. The distribution of reactive astrocytes was also evaluated by immunocytochemical localization of glial fibrillary acidic protein (GFAP). By 2 weeks postinjury, a prominent cavity was present in the frontopariental and occipital cortices. Although astrogliosis was most pronounced in the cortex surrounding the cavity, prominent reactive astrocytes were widely distributed throughout the injured hemisphere. This study characterized the pathological changes which occur after experimental traumatic brain injury. In particular, we propose that neuronal cell injury in the hippocampus serves as a useful ‘window’ to assess beneficial efficacy of pharmacological intervention in the treatment of brain injury.     

High concentrations of cannabinoids activate apoptosis in human U373MG glioma cells

Cannabinoids bind to two G-protein-coupled receptors, CB1 and CB2, expressed by neurons and cells of the immune system, respectively. Glioma cells (astrocyte-derived brain tumor cells) express cannabinoid receptors, and numerous in vitro and in vivo studies performed in rodents have concluded that apoptosis could be induced by cannabinoids in these cells. Whether this also applies to human cells is controversial; we, therefore, assessed the effect of cannabinoids on human glioma cell viability with the human astrocytoma cell line U373MG. We report here that U373MG human glioma cells are sensitive only to high concentrations of cannabinoids (>5 microg/ml for Delta(9)-THC). Similar concentrations of the compounds promoted a rapid activation of extracellular-regulated kinase and c-Jun NH2-terminal kinase, suggesting that cannabinoid receptors are functional in U373MG cells. Nevertheless, these kinases are not involved in cannabinoid-induced cell death in U373MG cells, insofar as blocking their activation with specific inhibitors does not reduce cell death. CB1 is expressed in U373MG cells and is involved in cannabinoid-induced cell death, in that blocking its activation with a specific antagonist (AM251) almost totally prevented cell death following incubation of the cells with Delta(9)-THC. In addition, as already reported, some cannabinoids may have modest proproliferative properties in U373MG cells. Human U373MG glioma cells are sensitive only to very high, pharmacologically irrelevant concentrations of cannabinoids, so it seems unlikely that cannabinoids would constitute promising molecules for treating malignant astrocytoma; they do not induce glioma cell death at doses that could be applied safely to humans.     

Application of a protein synthesis inhibitor into the ventral tegmental area,but not the nucleus accumbens,prevents behavioral sensitization to cocaine

Recent evidence implicates a crucial role for the ventral tegmental area (VTA) in the initiation of behavioral sensitization produced by repeated psychostimulant exposure, while changes in the nucleus accumbens (NAcc) are not critical during the initiation stage. We investigated whether the development of behavioral sensitization to repeated daily cocaine could be prevented by daily administration of the protein synthesis inhibitor, anisomycin, delivered onto VTA neurons. Rats were given five daily treatments as follows: obturators containing crystalline anisomycin or no compound (sham) were placed directly into the VTA 15 min prior to a saline (1 ml/kg, i.p.) or cocaine (15 mg/kg, i.p.) injection. After withdrawal for 8–9 days, the locomotor response to the same dose of saline or cocaine was monitored. No differences in the locomotor response to an acute saline challenge were found across the four groups. Animals given sham treatments in the VTA and daily cocaine demonstrated a significant augmentation in the locomotor response to a cocaine challenge compared to saline controls. Anisomycin treatments alone produced no effects on acute cocaine-induced locomotion. Further, a cocaine challenge in animals receiving daily anisomycin and cocaine elicited a non-augmented response similar to that of saline controls. Thus, the sensitized locomotor response to a cocaine challenge in daily cocaine pretreated animals was completely blocked by daily anisomycin treatment in the VTA. When daily anisomycin was administered into the NAcc along with daily cocaine, no blockade of behavioral sensitization was observed. These results provide support for a critical role of long-term changes in gene expression in the vicinity of VTA neurons mediating the development of sensitization to psychostimulants. © 1995 Wiley-Liss, Inc.     

Proliferative and migratory responses of astrocytes to in vitro injury

An in vitro "scratch-wound" model was used to evoke and investigate some astroglial responses to mechanical injury. The changes in the morphology, locomotion, and proliferation of injured astrocytes were analysed under culture conditions devoid of blood-derived cells responsible for activating the inflammatory cascade. The rate of proliferation was determined by immunocytochemical detection of BrdU-incorporating cells located next to or far from the wound. The motility of individual cells and the mass-advancement of cell-assemblies were monitored by computer controlled video-microscopy both in injured monolayers and in preparations of single cells or aggregates of astrocytes. The large sets of digitalized data allowed a reliable statistical evaluation of changes in cell positions providing a quantitative approach for studies on dynamics of cell locomotion. The results indicated that cultivated astrocytes respond to injury (1) with enhanced nestin immunoreactivity at the expanding processes, (2) with increased mitotic activity exceeding the rate caused by the liberation from contact inhibition, but (3) without specific, injury-induced activation of cell locomotion. Some advantages and drawbacks of "scratch-wound" models of astrocytic responses to mechanical injury are presented and discussed.     

Rho kinase activates ezrin-radixin-moesin (ERM) proteins and mediates their function in cortical neuron growth, morphology and motility in vitro

The ezrin-radixin-moesin (ERM) family of proteins contribute to cytoskeletal processes underlying many vital cellular functions. Their previously elucidated roles in non-neuronal cells are an indication of their potential importance in CNS neurons. The specific mechanisms of their activation are unknown, but are likely to depend on factors such as the cell type and biological context. For ERM proteins to become active they must be phosphorylated at a specific C-terminal threonine residue. In non-neuronal cells, several kinases, including the Rho GTPase family member Rho kinase, have been identified as capable of phosphorylating the C-terminal threonine. In these experiments we have investigated specifically the potential role of Rho kinase mediated ERM activation in cortical neurons, utilizing a new pharmacologic inhibitor of Rho kinase and quantitative analysis of aspects of neuronal functions potentially mediated by ERM proteins. Rho kinase inhibition significantly suppressed aspects of neuronal development including neurite initiation and outgrowth, as well as growth cone morphology, with a concomitant loss of phosphorylated ERM immunolabeling in areas associated with neuronal growth. The ability of the Rho kinase inhibitor to decrease the amount of pERM protein was shown by immunoblotting. Post-injury responses were negatively affected by Rho kinase inhibition, namely by a significant decrease in the number of regenerative neurites. We investigated a novel role for ERM proteins in neuron migration using a post-injury motility assay, where Rho kinase inhibition resulted in significant and drastic reduction in neuron motility and phosphorylated ERM immunolabeling. Thus, Rho kinase is an important activator of ERMs in mediating specific neuronal functions.     

Dynamic changes in the expression pattern of ecto-5'-nucleotidase in the rat model of cortical stab injury

Traumatic injury induces massive release of ATP in the extracellular space, where it influences numerous aspects of neuronal, astrocytic, and microglial responses to injury by activating P2X and P2Y receptors. The extracellular ATP actions are controlled by the ectonucleotidase enzyme pathway, which hydrolyses ATP to adenosine at all neuronal and nonneuronal cell types. Adenosine activates its P1 receptors, which have important neuroprotective roles. The rate-limiting enzyme in the ectonucleotidase pathway is ecto-5'-nucleotidase (e-5NT), which catalyzes the final step of dephosphorylation of AMP to adenosine. The aim of the present study was to characterize the expression pattern and cellular distribution of e-5NT in the perilesioned cortex at 4 hr and 1, 2, 7, and 15 days after unilateral cortical stab injury (CSI). Immunoblot and immunohistochemical studies showed that overall e-5NT expression was lower 4 hr and 1 day postinjury and then gradually increased above the control levels. Double-immunofluorescence studies further showed in control tissue the presence of the enzyme in the membranes surrounding neuronal somata and apical dendrites and less frequently in astrocytes. CSI caused a rapid (after 4 hr) and irreversible loss of the enzyme from neurons, accounting for a decrease in the overall enzyme expression. This was accompanied with a gradual increase in e-5NT-positive astrocytes, accounting for up-regulation of the enzyme levels in the injured area. Thus, CSI induced dynamic changes in the expression pattern of e-5NT that modify the ATP/adenosine ratio and the extent of P1 and P2 receptors activation and, therefore, outcome of the pathological processes after CSI.     

Effects of anisomycin on LTP in the hippocampal CA1: long-term analysis using optical recording

Long-term potentiation (LTP) in the hippocampal CA1 region and in the dentate gyrus consists of different stages: early LTP lasting minutes or several hours, and late LTP lasting longer than 4 h. It has been suggested that the late phase of LTP is dependent on protein synthesis. However, the experimental results of the effects of protein synthesis inhibitors are still confusing. We applied optical recording techniques to rat hippocampal slices, and re-evaluated the effects of a protein synthesis inhibitor, anisomycin, on LTP. Using a voltage-sensitive oxonol dye, NK3630 (RH482), LTP in the CA1 region could be monitored optically for a long-term period (7-8 h). In the presence of anisomycin, the potentiation of the EPSP (excitatory postsynaptic potential) lasted about 2-3 h, followed by a gradual decline in the signal amplitude. Statistically, significant effects of anisomycin were observed 6 h after LTP induction for 100 Hz tetanus and 8 h after LTP induction for 400 Hz tetanus. These results suggest that the early phase of LTP is independent of protein synthesis, while the late phase of potentiation (> 3-5 h) depends on protein synthesis.     

Juxtavascular microglia migrate along brain microvessels following activation during early postnatal development

Some parenchymal microglia in mammalian brain tissues, termed "juxtavascular microglia," directly contact the basal lamina of blood vessels; however, the functional consequences of this unique structural relationship are unknown. Here we used a rat brain slice model of traumatic brain injury to investigate the dynamic behavior of juxtavascular microglia following activation. Juxtavascular microglia were identified by confocal 3D reconstruction in tissue slices stained with a fluorescent lectin (FITC-IB4) that labels both microglia and blood vessel endothelial cells. Immunolabeling confirmed that juxtavascular cells were true parenchymal microglia (OX42+, ED2-) and not perivascular cells or pericytes. Time-lapse imaging in live tissue slices revealed that activating juxtavascular microglia withdraw most extant branches but often maintain contact with blood vessels, usually moving to the surface of a vessel within 1-4 h. Subsequently, some microglia migrate along the parenchymal surface of vessels, moving at rates up to 40 microm/h. Activated juxtavascular microglia sometimes repeatedly extend veil-like protrusions into the surrounding tissue, consistent with a role in tissue surveillance. Juxtavascular cells were twice as likely as nonjuxtavascular cells to be locomotory by 10 h in vitro, suggesting an enhanced activation response. Moreover, 38% of all juxtavascular cells migrated along a vessel, whereas this was never observed for a nonjuxtavascular cell. These observations identify a mobile subpopulation (10%-30%) of parenchymal microglia that activate rapidly and are preferentially recruited to the surfaces of blood vessels following brain tissue injury. The dynamic and sustained interaction of microglia with brain microvessels may facilitate signaling between injured brain parenchyma and components of the blood-brain barrier or circulating immune cells of the blood in vivo.     

Calpain activation and apoptosis in motor neurons of cultured adult mouse spinal cord

Calpain, a Ca2+-dependent cysteine protease, has been implicated in neuronal apoptosis following spinal cord injury. In this study, activation of calpain was investigated in motor neurons of adult spinal cord slices from the mouse, using a cell-permeable fluorogenic calpain substrate and Western blotting. Calpain was rapidly activated in the motor neurons of excised spinal cord slices and calpain activity was observed both in the cytoplasm and the nuclei. In these neurons, nuclear and chromatin condensation were pronounced. Both calpain inhibitor VI and EGTA (ethyleneglycol-bis(beta-aminoethyl ether) N' ,N' ,N' ,N' -tetraacetic acid) inhibited calpain activation and subsequent appearance of apoptotic nuclei. In contrast, the general caspase inhibitor Z-VAD.fmk had no effect. Calpain activation was also observed in the slices by Western blotting using an antibody to 150-kD calpain-cleaved alpha-fodrin fragment. These results show that calpain is rapidly activated in injured motor neurons and imply that this activation could be responsible for execution of caspase-independent apoptosis in injured adult motor neurons.     

Spinal Cord Compression Injury in Guinea Pigs: Structural Changes of Endothelium and Its Perivascular Cell Associations after Blood–Brain Barrier Breakdown and Repair

This study examines morphological changes of the blood–brain barrier (BBB) after spinal cord compression. The lowest thoracic segment (T13) of female guinea pigs was injured and the BBB was tested from 7 days to 5.5 months postinjury using intravenously injected horseradish peroxidase (HRP) as a tracer. Tracer leakage in the injured segment was verified with the light microscope and the fine structure of capillaries was examined. Diffuse tissue staining was observed at T13 up to 2 weeks following injury. A leaky BBB correlated with expected changes in the fine structure of endothelial cell junctions. These were predominantly nonoverlapping cell junctions which, in many instances, were separated by clefts between adjacent cells. At early survival times, numerous capillary profiles with juxtaposed astrocyte foot processes were noted in addition to altered cell associations. Complete sealing of the BBB against interstitial HRP leakage was not observed until 17 days postinjury. After the first week, some of the endothelial cells were contacted by macrophages, processes of perivascular microglia, and processes of swollen and degenerating astrocytes. Perivascular spaces varied in extent and contained amorphous deposits of extracellular materials in addition to supernumerary layers of basal lamina. The early changes were followed by profound tissue restructuring due to loss of both neurons and glia. At longer survival times the BBB to HRP repaired. Endothelial cells formed complex overlapping junctions with zonulae occludentes. Most of the capillaries in the injured segment were no longer in direct contact with astrocyte foot processes, although reactive astrocytes constituted the predominant cell type in the remaining gray matter. Substantial expansion of perivascular spaces was evident. The cytoplasm of endothelial cells had numerous pinocytotic vesicles. Perivascular spaces contained layers of assembled collagen arranged perpendicularly to each other in addition to amorphous matrix materials. The findings suggest that decoupling of astrocyte foot processes from endothelial cell surfaces does not prevent reformation of tight junctions. It remains to be examined what effects the larger perivascular spaces, extracellular matrix deposits, and changes of cell associations may have on transport systems and ionic buffering. The data are relevant for estimating an opportune time for application of barrier-impermeable drugs to the lesion area.     

Cell-specific activation of RIPK1 and MLKL after intracerebral hemorrhage in mice

Receptor-interacting protein kinase-1 (RIPK1) is a master regulator of cell death and inflammation, and mediates programmed necrosis (necroptosis) via mixed-lineage kinase like (MLKL) protein. Prior studies in experimental intracerebral hemorrhage (ICH) implicated RIPK1 in the pathogenesis of neuronal death and cognitive outcome, but the relevant cell types involved and potential role of necroptosis remain unexplored. In mice subjected to autologous blood ICH, early RIPK1 activation was observed in neurons, endothelium and pericytes, but not in astrocytes. MLKL activation was detected in astrocytes and neurons but not endothelium or pericytes. Compared with WT controls, RIPK1 kinase-dead (RIPK1^D138N/D138N) mice had reduced brain edema (24 h) and blood-brain barrier (BBB) permeability (24 h, 30 d), and improved postinjury rotarod performance. Mice deficient in MLKL (Mlkl^-/-) had reduced neuronal death (24 h) and BBB permeability at 24 h but not 30d, and improved post-injury rotarod performance vs. WT. The data support a central role for RIPK1 in the pathogenesis of ICH, including cell death, edema, BBB permeability, and motor deficits. These effects may be mediated in part through the activation of MLKL-dependent necroptosis in neurons. The data support development of RIPK1 kinase inhibitors as therapeutic agents for human ICH.     

Cortical expression of nuclear factor kappaB after human brain contusion

The aim of current study was to analyze the binding activity and the temporal and cellular expression of nuclear factor kappa B (NF-kappaB) in human contused brain. Eighteen contused brain samples were obtained from 17 patients undergoing surgery for brain contusions 5-80 h after trauma. NF-kappaB binding activity was detected by electrophoretic mobility shift assay (EMSA), and temporal and cellular expression of NF-kappaB subunits p65 and p50 was analyzed by immunohistochemistry. The results showed that a progressive upregulation of NF-kappaB activity occurred in the area surrounding the injured brain with the time from brain trauma to operation. The maximal expression of NF-kappaB was detected after 48 h postinjury. The expression of NF-kappaB p65 was mainly located at glial and vascular endothelial cells without expression at neurons. The expression of NF-kappaB p50 was mainly located at glial cells, a little at neurons and no expression at vascular endothelial cells. Within 24 h postinjury, both NF-kappaB p65 and p50 immunoreactivity was mainly observed in the nucleus of cells. After 24 h postinjury, NF-kappaB p65 labeling was found in the both nucleus and cytoplasm of glial and endothelial cells; otherwise, p50 labeling was primarily found in the nucleus of glial cells and in the nucleus, cytoplasm and process of neurons. It is concluded that NF-kappaB could be highly upregulated at human contused brain and the cellular pattern of p65 and p50 expression might be closely associated with the cell functions.