Neutrophil Infiltration and Matrix Metalloproteinase-9 in Lacunar Infarction.

We use the modified pial vessel disruption rat model to elucidate the cellular and molecular mechanisms of cavitation as it plays a role in lacunar infarction. Here we discuss the similarities between the genesis of pulmonary cavitation in various animal models and lacunar infarction in the cerebral cortex of rats. Both pathological processes involve the creation of a cavity surrounded by fibroblasts or reactive astrocytes. A crucial step in both, the lung and the cerebral cortex, appears to be the migration of neutrophils across the endothelial barrier into the parenchyma. In the lung and cerebral cortex this involves release of matrix metalloproteinase-9 (MMP-9). Inside the parenchyma neutrophils continue to release MMP-9. In both situations batimastat (BB-94) and minocycline reduce release of MMP-9 and prevent cavitation. In the cerebral cortex MMP-9 release by resident microglia plays an additional role. We therefore advance the hypothesis that cavitation in both tissues is driven by MMP-9 originating from invading neutrophils. Therapeutic intervention has to focus on these blood-borne intruder cells and specific MMP actions. Batimastat and its derivatives (marimastat, BB-1101, mCGS-27023-A, ilomastat, GM6001, CTK8G1150) are already in clinical or experimental use in humans for anti-cancer treatment, and these clinically relevant drugs could be repurposed to act as anti-inflammatory to counter neutrophil contribution to lung or cerebral cortex cavitation.

Prolonged adenosine A1 receptor activation in hypoxia and pial vessel disruption focal cortical ischemia facilitates clathrin-mediated AMPA receptor endocytosis and long-lasting synaptic inhibition in rat hippocampal CA3-CA1 synapses: differential regulation of GluA2 and GluA1 subunits by p38 MAPK and JNK.

Activation of presynaptic adenosine A1 receptors (A1Rs) causes substantial synaptic depression during hypoxia/cerebral ischemia, but postsynaptic actions of A1Rs are less clear. We found that A1Rs and GluA2-containing AMPA receptors (AMPARs) form stable protein complexes from hippocampal brain homogenates and cultured hippocampal neurons from Sprague Dawley rats. In contrast, adenosine A2A receptors (A2ARs) did not coprecipitate or colocalize with GluA2-containing AMPARs. Prolonged stimulation of A1Rs with the agonist N(6)-cyclopentyladenosine (CPA) caused adenosine-induced persistent synaptic depression (APSD) in hippocampal brain slices, and APSD levels were blunted by inhibiting clathrin-mediated endocytosis of GluA2 subunits with the Tat-GluA2-3Y peptide. Using biotinylation and membrane fractionation assays, prolonged CPA incubation showed significant depletion of GluA2/GluA1 surface expression from hippocampal brain slices and cultured neurons. Tat-GluA2-3Y peptide or dynamin inhibitor Dynasore prevented CPA-induced GluA2/GluA1 internalization. Confocal imaging analysis confirmed that functional A1Rs, but not A2ARs, are required for clathrin-mediated AMPAR endocytosis in hippocampal neurons. Pharmacological inhibitors or shRNA knockdown of p38 MAPK and JNK prevented A1R-mediated internalization of GluA2 but not GluA1 subunits. Tat-GluA2-3Y peptide or A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine also prevented hypoxia-mediated GluA2/GluA1 internalization. Finally, in a pial vessel disruption cortical stroke model, a unilateral cortical lesion compared with sham surgery reduced hippocampal GluA2, GluA1, and A1R surface expression and also caused synaptic depression in hippocampal slices that was consistent with AMPAR downregulation and decreased probability of transmitter release. Together, these results indicate a previously unknown mechanism for A1R-induced persistent synaptic depression involving clathrin-mediated GluA2 and GluA1 internalization that leads to hippocampal neurodegeneration after hypoxia/cerebral ischemia.

Involvement of matrix metalloproteinases-2 and -9 in the formation of a lacuna-like cerebral cavity.

We used a modified pial vessel disruption (PVD) protocol with adult male Wistar rats to mimic small-vessel stroke in the cerebral cortex. Within 3 weeks, this lesion develops into a single lacuna-like cavity, which is fluid-filled and encapsulated by reactive astrocytes. Minocycline treatment that commences 1 hr after lesion and continues for 6 days prevents the cavitation and causes a filling of the lesion with reactive astrocytes and no barrier. Here, we determined whether inhibition of matrix metalloproteinases-2 and -9 (MMPs) mediates this minocycline action. Confocal microscopy revealed increased punctate staining of MMPs inside the lesion sites after 2 days of PVD. Astrocytes lined the lesion border but showed sparse localization inside the lesion. In contrast, increased MMP levels inside the lesion coincided with increased ED1 or OX-42 immunostaining, suggesting that MMP elevation reflected increased secretions from microglia/macrophages. Imaging analyses also revealed that minocycline administered for 2 days before animal euthanasia, significantly decreased MMP levels within the lesion. Moreover, Western blot analysis of cortical tissue extracts showed a significant 30-40% upregulation of MMPs 2 days after lesion. Minocycline administered 2 hr before the lesion significantly inhibited both MMP-9 and MMP-2 levels by ∼40%. In contrast, minocycline administered 1 hr after the lesion only decreased MMP-9 levels by ∼30%. Because MMP inhibition with batimastat injection also prevented cavity formation at 21 days, we conclude that minocycline prevented the creation of a lacuna-like cyst in the cerebral cortex by inhibiting the MMP secretion from microglia in the affected tissue.

In the mouse cortex, oligodendrocytes regain a plastic capacity, transforming into astrocytes after acute injury.

Acute brain injuries evoke various response cascades directing the formation of the glial scar. Here, we report that acute lesions associated with hemorrhagic injuries trigger a re-programming of oligodendrocytes. Single-cell RNA sequencing highlighted a subpopulation of oligodendrocytes activating astroglial genes after acute brain injuries. By using PLP-DsRed1/GFAP-EGFP and PLP-EGFPmem/GFAP-mRFP1 transgenic mice, we visualized this population of oligodendrocytes that we termed AO cells based on their concomitant activity of astro- and oligodendroglial genes. By fate mapping using PLP- and GFAP-split Cre complementation and repeated chronic in vivo imaging with two-photon laser-scanning microscopy, we observed the conversion of oligodendrocytes into astrocytes via the AO cell stage. Such conversion was promoted by local injection of IL-6 and was diminished by IL-6 receptor-neutralizing antibody as well as by inhibiting microglial activation with minocycline. In summary, our findings highlight the plastic potential of oligodendrocytes in acute brain trauma due to microglia-derived IL-6.

Doublecortin-expressing cells in the ischemic penumbra of a small-vessel stroke.

A cortical lesion was induced by disrupting the medium-size pial vessels, which led to a cone-shaped cortical lesion and turned into a fluid-filled cavity surrounded by a glial acidic fibrillary protein-positive (GFAP(+)) glia limitans 21 days after injury. Therefore, it mimics conditions of lacunar infarctions, one of the most frequent human stroke pathologies. Doublecortin (DCX)-positive cells were present in the neocortex and corpus callosum at the base of the lesion. The number of DCX-positive cells in the corpus callosum was significantly increased from day 5 to day 14 compared with the control group. In contrast, there were no DCX-positive cells in neocortex of control animals; the DCX-positive cells appeared in the neocortex after lesioning and were maintained until 14 days postlesioning. Some of the DCX-positive cells were also immunoreactive for beta III-tubulin, another marker of immature neurons. They did not stain positively for markers of glia cells. The presence of these DCX-positive cells near the lesion might indicate a migratory pathway for developing neuroblasts from the subventricular zone (SVZ) through the corpus callosum to the lesion. SVZ cells were labeled with a lipophilic molecule, 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) stereotaxical injections. Although rostral migratory stream and olfactory bulb were intensely labeled, no CFSE-containing cells were found in the cortex beneath the lesion. These results do not support the idea that the DCX-positive cells were originating from neural precursors of the SVZ, but they might be generated from local progenitor cells.

The need for animal models in small-vessel brain disease.

An argument is made that small-vessel stroke, which usually results in lacunar infarction, is a serious medical problem. Therefore, it is surprising that only a few animal models exist that mimic small-vessel stroke and that these models have not been used for a systematic investigation of the genesis of lacunar infarctions. We make a case that the modified pial vessel class II disruption model mimics certain important aspects of lacunar infarctions, namely cavitation caused specifically by ischemia of smaller vessels. We found evidence that upregulation of inflammatory properties within a few days of inducing lesions prevents repopulation of the lesion with reactive astrocytes. We propose that this is the key mechanism by which cavitation occurs weeks later. We also found that treatment with minocycline after induction of lesions but before cavitation prevented the formation of the fluid-filled cavity. Rather than being walled off, the lesion apparently became part of the brain parenchyma and consisted of reactive astrocytes. We conclude that this new model can be used to investigate the mechanism of lacune formation and its prevention.

Minocycline treatment prevents cavitation in rats after a cortical devascularizing lesion.

Minocycline, a second-generation tetracycline, has been shown to possess neuroprotective effects in animal models of stroke. Pial vessel disruption in adult Wistar rats leads to a cone-shaped cortical lesion and turns into a fluid-filled cavity surrounded by a GFAP+ glia limitans 21 days after injury. This mimics the clinical situation in lacunar infarcts. Minocycline was given intraperitoneally at a dose of 45 mg/kg 1 and 12 h after lesioning, followed by 22.5 mg/kg twice daily until 6 days after lesioning. Control rats received intraperitoneal injections of equivalent volumes of saline. Cavitation was prevented in five out of six minocycline-treated animals and the glia limitans did not appear as the space was filled with GFAP+ reactive astrocytes. However, the number of activated microglia showed no difference between minocycline-treated and -untreated groups. Minocycline did not reduce the number of infiltrating leukocytes, predominately polymorphonuclear neutrophils (PMNs) determined by myeloperoxidase immunoreactivity, or infiltration of CD3+ lymphocytes. The pial vessel occlusion induced a significant upregulation of IL-1beta expression; however, minocycline treatment did not significantly alter this upregulation of IL-1beta. In this study, we found minocycline facilitated the repopulation of the lesion by reactive astrocytes and therefore prevented cavitation; however, we could not identify the molecular signal.

Vimentin-expressing proximal reactive astrocytes correlate with migration rather than proliferation following focal brain injury.

Vimentin-expressing astrocytes in the adult are commonly associated with the proximal, most reactive gliotic response ultimately leading to the formation of a new glial limitans. It was thought, since vimentin expression and astroglial proliferation are most prominent nearest the lesion site, that vimentin may be a characteristic of immature newly divided astrocytes. We recently established a unique distribution of vimentin-expressing reactive astrocytes at the base of a focal cortical ischemic lesion in rats. The purpose of the present study was to assess the correlation of proliferation and migration with this unique distribution following focal injury. With the use of bromodeoxyuridine (BrdU) and immunohistochemistry for astrocytes and microglia/macrophages, proliferation and migration of cells was shown to be throughout the ipsilateral hemisphere on day one and become progressively more centralized to the lesion by day 3. The vimentin-expressing area at the base of the lesion does not exhibit distinguishable proliferation rates from non-vimentin-expressing regions surrounding the lesion and did not demonstrate obvious double labeling with BrdU+ cells, although on occasion vimentin expression is closely associated with BrdU. However, this region did become a focal point for migration into and around the lesion by day 3. Additionally, asymmetrical distribution of vimentin was shown in four different injury models with vimentin+ cells always situated between the lesion and the corpus callosum. It is concluded that although vimentin-expressing cells did not correlate with proliferating cells in these focal injury models, perhaps this distinct population of reactive astrocytes serve as a source of cytokines or as a physical conduit for migrating cells from distant sites through the corpus callosum.

Inhibition of brain swelling after ischemia-reperfusion by ß-adrenergic antagonists: correlation with increased K+ and decreased Ca2+ concentrations in extracellular fluid.

Infarct size and brain edema following ischemia/reperfusion are reduced by inhibitors of the Na+, K+, 2Cl-, and water cotransporter NKCC1 and by ß1-adrenoceptor antagonists. NKCC1 is a secondary active transporter, mainly localized in astrocytes, driven by transmembrane Na+/K+ gradients generated by the Na+,K+-ATPase. The astrocytic Na+,K+-ATPase is stimulated by small increases in extracellular K+ concentration and by the ß-adrenergic agonist isoproterenol. Larger K+ increases, as occurring during ischemia, also stimulate NKCC1, creating cell swelling. This study showed no edema after 3 hr medial cerebral artery occlusion but pronounced edema after 8 hr reperfusion. The edema was abolished by inhibitors of specifically ß1-adrenergic pathways, indicating failure of K+-mediated, but not ß1-adrenoceptor-mediated, stimulation of Na+,K+-ATPase/NKCC1 transport during reoxygenation. Ninety percent reduction of extracellular Ca2+ concentration occurs in ischemia. Ca2+ omission abolished K+ uptake in normoxic cultures of astrocytes after addition of 5 mM KCl. A large decrease in ouabain potency on K+ uptake in cultured astrocytes was also demonstrated in Ca2+-depleted media, and endogenous ouabains are needed for astrocytic K+ uptake. Thus, among the ionic changes induced by ischemia, the decrease in extracellular Ca2+ causes failure of the high-K+-stimulated Na+,K+-ATPase/NKCC1 ion/water uptake, making ß1-adrenergic activation the only stimulus and its inhibition effective against edema.

Potassium homeostasis in the ischemic brain.

Extracellular [K+] can range within 2.5-3.5 mM under normal conditions to 50-80 mM under ischemic and spreading depression events. Sustained exposure to elevated [K+]o has been shown to cause significant neuronal death even under conditions of abundant glucose supply. Astrocytes are well equipped to buffer this initial insult of elevated [K] through extensive gap junctional coupling, Na+/K+ pump activity (with associated glycogen and glycolytic potential), and endfoot siphoning capability. Their abundant energy availability and alkalinizing mechanisms help sustain Na+/K+ ATPase activity under ischemic conditions. Furthermore, passive K+ uptake mechanisms and water flux mediated through aquaporin-4 channels in endfoot processes are important energy-independent mechanisms. Unfortunately, as the length of ischemic episode is prolonged, these mechanisms increase to a point where they begin to have repercussions on other important cellular functions. Alkalinizing mechanisms induce an elevation of [Na+]i, increasing the energy demand of Na+/K+ ATPase and leading to eventual detrimental reversal of the Na+/glutamate- cotransporter and excitotoxic damage. Prolonged ischemia also results in cell swelling and activates volume regulatory processes that release excessive excitatory amino acids, further exacerbating excitotoxic injury. In the days following ischemic injury, reactive astrocytes demonstrate increased cell size and process thickness, leading to improved spatial buffering capacity in regions outside the lesion core where there is better neuronal survival. There is a substantial heterogeneity among reactive astrocytes, with some close to the lesion showing decreased buffering capacity. However, it appears that both Na+/K+ ATPase activity (along with energy production processes) as well as passive K+ uptake mechanisms are upregulated in gliotic tissue outside the lesion to enhance the above-mentioned homeostatic mechanisms.

Complex expression and localization of inactivating Kv channels in cultured hippocampal astrocytes.

Voltage-gated potassium channels are well established as critical for setting action potential frequency, membrane potential, and neurotransmitter release in neurons. However, their role in the "nonexcitable" glial cell type is yet to be fully understood. We used whole cell current kinetics, pharmacology, immunocytochemistry, and RT-PCR to characterize A-type current in hippocampal astrocyte cultures to better understand its function. Pharmacological analysis suggests that approximately 70, 10, and <5% of total A current is associated with Kv4, Kv3, and Kv1 channels, respectively. In addition, pharmacology and kinetics provide evidence for a significant contribution of KChIP accessory proteins to astrocytic A-channel composition. Localization of the Shaw Kv3.4 channel to astrocytic processes and the Shal Kv4.3 channel to soma suggest that these channels serve a specific function. Given this complex A-type channel expression pattern, we assessed the role of A currents in membrane voltage oscillations in response to current injections. Although TEA-sensitive delayed-rectifying currents are involved in the extent of repolarization, 4-AP-sensitive A currents serve to increase the rate. As in neurons, this effect may enable astrocytes to respond rapidly to high-frequency synaptic events. Our results indicate that hippocampal astrocytes in vitro express multiple A-type Kv channel alpha-subunits with accessory, possibly Ca(2+)-sensitive, cytoplasmic subunits that appear to be specifically localized to subcellular membrane compartments. Function of these channels remains to be determined in a physiological setting. However, this study suggests that they enable astrocytes to respond rapidly with membrane voltage oscillations to high-frequency incoming signals, possibly synchronizing astrocyte function to neuronal activity.

Chloride/anion channels in glial cell membranes.

At least seven different chloride/anion currents have now been identified in astrocytes, oligodendrocytes/Schwann cells, and microglia. Only for two of these currents is the corresponding gene known. One of these genes is not encoding for a chloride channel, but for a class of mitochondria-like pores also found in cell membranes. Astrocytes and oligodendrocytes differ in their resting properties: astrocytes accumulate chloride but do not have a significant permeability. Oligodendrocytes have a close to passive distribution and a significant permeability. Under certain circumstances, astrocytes can express a resting chloride conductance. Reactive and neoplastic astrocytes as well as astrocytes with an altered shape exhibit a resting conductance. The function of these channels certainly involves volume regulation. Other possible functions are potassium homeostasis, migration, proliferation (in microglia), and involvement in spreading depression waves. Of greatest interest are two phenomena discovered in situ: The ClC-2 channel is only found in astrocytic endfeet near blood capillaries adjacent to neuronal GABA(A) receptors. In the supraoptic nucleus of the hypothalamus, there is an osmosensitive astrocytic taurine release. This released taurine interacts with glycine receptors in neighboring neurons, causing inhibition. It is assumed that with the future availability of more in situ, rather than in vitro, studies, an increased number of such complex interactions between glial cells, neurons, and blood vessels will be discovered.

Unusual topographical pattern of proximal astrogliosis around a cortical devascularizing lesion.

Class II vessels were disrupted on the cortical surface of adult rats within a circular 5-mm-diameter area. This consistently resulted in the formation of a conical lesion by day 1, with a cystic cavity forming by day 21. Four markers were used to identify the glial response surrounding the lesion. The antibody used against S100beta marked the largest astrocytic pool in the gray matter of the cerebral cortex; only approximately 5% of astrocytes were glial fibrillary acidic protein (GFAP)(+) in control animals. GFAP served as a marker for distal reactive gliosis and vimentin (VIM) for proximal gliosis. Isolectin B4 was used as an additional marker to distinguish VIM(+) microglia from astrocytes inside the lesion area. Three immunohistochemically distinct areas of reactive astrocytes surrounding the lesion were found within 24 hr of injury and lasted through day 6. The first area, in contrast to focal traumatic injuries, consisted of a 196-microm-thick boundary layer of S100beta(+) cells immediately surrounding the lesion that never expressed GFAP or VIM by day 6. This boundary layer turns into a GFAP(+) glial limitans encasing the cystic cavity by day 21. A second unusual extended area around the base of the lesion reaching partly into the corpus callosum consisted of S100beta(+)/GFAP(+)/VIM(+) cells. This region appears to be compatible with the local or proximal gliotic response usually found completely surrounding other focal-type injuries. The proximal response at the base of the lesion developed over the first 3 days in the following sequence: S100beta(+)/GFAP(-)/VIM(-) to S100beta(+)/GFAP(+)/VIM(-) to S100beta(+)/GFAP(+)/VIM(+). Ninety percent of the astrocytes in this area express VIM. This is very high compared with findings in stab-wound preparations, where only 10% of astrocytes (surrounding entire lesion) are found to be VIM(+). A third region, consistent with a remote or distal reactive gliotic response, demonstrated staining for S100beta and had increased GFAP contents throughout the neocortical hemisphere. Cells in this region were never found to be VIM(+). Among S100beta(+) cells close to the boundary region, more than 80% expressed detectable GFAP by 2 days after lesioning. S100beta(+) cells 1 mm more laterally (distal to lesion) did not express GFAP to the same level until day 6. Thus, we find three immunohistochemically distinct populations of reactive astrocytes surrounding the focal ischemic lesion. In contrast to the case for stab-wound traumatic injury, the response closest to and surrounding the lesion did not up-regulate GFAP or VIM by day 6. The proximal response was, instead, more remote and only at the base of the lesion, extending partly into the corpus callosum.

Intracellular chloride modulates A-type potassium currents in astrocytes.

Application of the GABA(A) receptor agonist muscimol to astrocytes in situ or in vitro results in a receptor-mediated Cl(-) current with a concomitant block of outward K(+) currents. The effect on K(+) current is largely selective for the inactivating A-type current. Parallel experiments with various Cl(-) pipette concentrations show a significant reduction in A-type current under low Cl(-) conditions with minimal effect on delayed current. In addition, lower Cl(-) conditions caused a depolarizing shift of steady-state inactivation (V(1/2), -68 to -57 mV) and activation (V(1/2), -5.8 to 34 mV) kinetics of A-type current only. Cl(-) had no effect on the time course of inactivation or reactivation kinetics, suggesting the Cl(-)-mediated effect is largely on activation kinetics, indirectly affecting steady-state inactivation. Muscimol application to astrocytes under perforated patch control (gramicidin) displayed a similar block of A-type current to that of conventional whole cell patch at 40 or 20 mM pipette Cl(-) concentrations. With barium application under perforated patch conditions, the study of muscimol-mediated Cl(-) current in isolation of the effect on K(+) currents was possible. This allowed estimation of intracellular Cl(-) concentration from receptor current reversal information. The average intracellular Cl(-) concentration was found to be 29 +/- 3.2 mM. The effect on activation kinetics and lack of effect on time course of inactivation or reactivation suggest that intracellular anion concentrations have an effect on the K(+) channel voltage sensor region. Cl(-) may modulate K(+) currents by altering membrane field potentials surrounding K(+) channel proteins.

pCLCA1 becomes a cAMP-dependent chloride conductance mediator in Caco-2 cells.

Members of the CLCA protein family are expressed in airway and intestinal epithelium, where they may participate in secretory activity as mediators of chloride conductance. A calcium-dependent chloride conductance has been observed upon expression of CLCA proteins in non-epithelial cell lines. The pCLCA1 gene, cloned in our laboratory, codes for a product containing a unique A-kinase consensus acceptor site not found in other CLCA proteins. Calcium-dependent, but not cAMP-dependent, chloride conductance increased when pCLCA1 was expressed in NIH/3T3 fibroblasts. We transfected the Caco-2 human colon carcinoma cell line with pCLCA1 to investigate the regulation of CLCA-associated chloride conductance in this differentiated epithelial cell line. Expression of pCLCA1 in the Caco-2 cell line enhanced cAMP-responsive 36Cl efflux, short circuit current, and whole cell chloride current in these cells. This cAMP-dependent chloride conductance was localized to the apical membrane of polarized Caco-2 cells.

pCLCA1 lacks inherent chloride channel activity in an epithelial colon carcinoma cell line.

The effects of CLCA protein expression on the regulation of Cl(-) conductance by intracellular Ca(2+) and cAMP have been studied previously in nonepithelial cell lines chosen for low backgrounds of endogenous Cl(-) conductance. However, CLCA proteins have been cloned from, and normally function in, differentiated epithelial cells. In this study, we examine the effects of differentiation of the Caco-2 epithelial colon carcinoma cell line on modulation of Cl(-) conductance by pCLCA1 protein expression. Cl(-) transport was measured as (36)Cl(-) efflux, as transepithelial short-circuit currents, and as whole cell patch-clamp current-voltage relations. The rate of (36)Cl(-) efflux and amplitude of currents in patch-clamp studies after the addition of the Ca(2+) ionophore A-23187 were increased significantly by pCLCA1 expression in freshly passaged Caco-2 cells. However, neither endogenous nor pCLCA1-dependent Ca(2+)-sensitive Cl(-) conductance could be detected in 14-day-postpassage cells. In contrast to Ca(2+)-sensitive Cl(-) conductance, endogenous cAMP-dependent Cl(-) conductance does not disappear on Caco-2 differentiation. cAMP-dependent Cl(-) conductance was modulated by pCLCA1 expression in Caco-2 cells, and this modulation was observed in freshly passaged and in mature 14-day-postpassage Caco-2 cultures. pCLCA1 mRNA expression, antigenic pCLCA1 protein epitope expression, and pCLCA1 function as a modulator of cAMP-dependent Cl(-) conductance were retained through differentiation in Caco-2 cells, whereas Ca(2+)-dependent Cl(-) conductance disappeared. We conclude that pCLCA1 expression may increase the sensitivity of preexisting endogenous Cl(-) channels to Ca(2+) and cAMP agonists but apparently lacks inherent Cl(-) channel activity under growth conditions where endogenous channels are not expressed.