Nuclear translocation of endonuclease G in degenerating neurons after permanent middle cerebral artery occlusion in mice.

Endonuclease G (EndoG) is a mitochondrial enzyme, known to be involved in caspase-independent cell death following translocation to the cellular nucleus. Nuclear translocation of EndoG has been observed in the ischemic area following transient occlusion of the middle cerebral artery (MCA) in mice, but not after permanent MCA occlusion. In this study we investigated the cellular and temporal expression of EndoG in infarcted cortex during the first 24 h after permanent MCA occlusion in mice, using immunohistochemistry, quantitative rt-PCR and cell specific immunoflourescence markers. EndoG translocated from the cytoplasm to the nucleus as early as 4 h and with a significant increase in the number of EndoG positive nuclei at 12 and 24 h after MCA occlusion. Nuclear translocation of EndoG was observed in degenerating NeuN positive neurons that were evenly distributed throughout the developing infarct. Translocation of EndoG was supported by unaltered EndoG mRNA levels. EndoG was neither expressed in GFAP positive astrocytes nor in CD11b positive microglia/macrophages. In contrast, CD11b positive microglia, but not infiltrating CD11b positive bone marrow-derived macrophages, were shown to express activated caspase-3. The translocation of EndoG to the nucleus of neurons in the infarct implicates EndoG in ischemic neuronal degeneration after permanent MCA occlusion in mice. Increased knowledge about EndoG involvement in ischemic neuronal cell death in mice might offer a promise to control processes involved in neuronal cell death pathways in stroke.

Axonal lesion-induced microglial proliferation and microglial cluster formation in the mouse.

Microglia are innate immune cells and form the first line of defense of the CNS. Proliferation is a key event in the activation of microglia in acute pathology, and has been extensively characterized in rats, but not in mice. In this study we investigated axonal-lesion-induced microglial proliferation and surface antigen expression in C57BL/6 mice. Transection of the entorhino-dentate perforant path projection results in an anterograde axonal and a dense terminal degeneration that induces a region-specific activation of microglia in the dentate gyrus. Time-course analysis showed activation of microglial cells within the first week post-lesion and cell counting demonstrated a significant 1.6-fold increase in microglial numbers 24 h post-lesion reaching a maximal 3.8-fold increase 3 days post-lesion compared with controls. Double staining for the microglial macrophage antigen-1 and the proliferation marker bromodeoxyuridine, injected 1 h prior to perfusion, showed that lesion-reactive microglia accounted for the vast majority of proliferating cells. Microglia proliferated as soon as 24 h after lesion and 25% of all microglial cells were proliferating 3 days post-lesion. Immunofluorescence double staining showed that most activated, proliferating microglia occurred in multicellular clusters and co-expressed the intercellular adhesion molecule-1 and the hematopoietic stem cell marker cluster of differentiation 34. In conclusion, this study extends observations of axonal lesion-induced microglial proliferation in rats to mice, and provides new information on early microglial proliferation and microglial cluster formation and surface antigen expression in the mouse.

Microglia and macrophages express tumor necrosis factor receptor p75 following middle cerebral artery occlusion in mice.

The proinflammatory and potential neurotoxic cytokine tumor necrosis factor (TNF) is produced by activated CNS resident microglia and infiltrating blood-borne macrophages in infarct and peri-infarct areas following induction of focal cerebral ischemia. Here, we investigated the expression of the TNF receptors, TNF-p55R and TNF-p75R, from 1 to 10 days following permanent occlusion of the middle cerebral artery in mice. Using quantitative polymerase chain reaction (PCR), we observed that the relative level of TNF-p55R mRNA was significantly increased at 1-2 days and TNF-p75R mRNA was significantly increased at 1-10 days following arterial occlusion, reaching peak values at 5 days, when microglial-macrophage CD11b mRNA expression was also increased. In comparison, the relative level of TNF mRNA was significantly increased from 1 to 5 days, with peak levels 1 day after arterial occlusion. In situ hybridization revealed mRNA expression of both receptors in predominantly microglial- and macrophage-like cells in the peri-infarct and subsequently in the infarct, and being most marked from 1 to 5 days. Using green fluorescent protein-bone marrow chimeric mice, we confirmed that TNF-p75R was expressed in resident microglia and blood-borne macrophages located in the peri-infarct and infarct 1 and 5 days after arterial occlusion, which was supported by Western blotting. The data show that increased expression of the TNF-p75 receptor following induction of focal cerebral ischemia in mice can be attributed to expression in activated microglial cells and blood-borne macrophages.

A quantitative in situ hybridization and polymerase chain reaction study of microglial-macrophage expression of interleukin-1beta mRNA following permanent middle cerebral artery occlusion in mice.

Interleukin-1beta (IL-1beta) is known to play a central role in ischemia-induced brain damage in rodents. In comparison to the rat, however, the available data on the cellular synthesis of IL-1beta mRNA and protein in the mouse are very limited. Here, we report on the time profile, the topography and the quantitative, cellular expression of IL-1beta mRNA in mice subjected to permanent occlusion of the distal middle cerebral artery (MCA). The in situ hybridization analysis showed that IL-1beta mRNA was expressed during the first post-surgical hour in a small number of high-expressing macrophage-like cells, located in cortical layers I and II of the future infarct. At 2 h, a significant number of faintly labeled IL-1beta mRNA-expressing cells had appeared in the developing peri-infarct, and the number remained constant at 4 h and 6 h, when the hybridization signal began to distribute to the cellular processes. Quantitative PCR performed on whole hemispheres showed a significant 20-fold increase in the relative level of IL-1beta mRNA at 12 h and a highly significant 42-fold increase at 24 h, at which time single IL-1beta mRNA-expressing cells were supplemented by aggregates and perivascular infiltrates of intensely labeled IL-1beta mRNA-expressing cells. Immunohistochemistry and double immunohistochemical stainings in addition to combined in situ hybridization, confirmed that the intensely labeled IL-1beta mRNA-expressing and IL-1beta protein synthesizing cells predominantly were glial fibrillary acidic protein-immunonegative, macrophage associated antigen-1-immunopositive microglia-macrophages. By day 5 there was a dramatic decline in the relative level of IL-1beta mRNA in the ischemic hemisphere. In summary, the data provide evidence that permanent occlusion of the distal MCA in mice results in expression of IL-1beta mRNA and IL-1beta synthesis in spatially and temporally segregated subpopulations of microglia and macrophages.

Non-invasive method for sampling and extraction of mouse DNA for PCR.

We adapted a non-invasive, fast, reliable and inexpensive procedure for the sampling and extraction of deoxyribonucleic acid (DNA) for genetic testing of mice. The procedure is based on a simple DNA extraction procedure used in the forensic genetic testing of humans. It involves mouth swabbing of the inner cheek using a cotton stick, followed by alkaline lysis of the harvested buccal epithelial cells. This procedure allows for repeated sampling and genetic testing of the individual mouse, and it is faster, simpler and, in our hands, more reliable than the currently used routine procedures for the sampling and extraction of mouse DNA. Current procedures all involve biopsy of a piece of the tail, ear or toe, followed by lengthy procedures to release and isolate the DNA.

A specific and sensitive method for visualization of tumor necrosis factor in the murine central nervous system.

We present here sensitive, simple and robust methods for detection of tumor necrosis factor (TNF) mRNA and TNF in histological sections and homogenates of brain tissue from mice subjected to focal cerebral ischemia or hippocampal axonal lesioning. Both types of lesions are characterized by induction of TNF synthesis in resident microglial cells, which in the ischemic lesions are supplemented by TNF synthesizing, blood-borne macrophages. In situ hybridization for TNF mRNA is performed using alkaline phosphatase-labelled oligodeoxynucleotide probes. These probes show excellent rendition of individual cells, and can successfully be combined with immunohistochemical procedures. We also describe a sensitive immunohistochemical method for detection of TNF, which can be combined with visualization of an additional antigen. The specificity of the histological procedures are confirmed by RT-PCR and Western blot analysis on homogenates prepared from microdissected brain regions. Advantages and disadvantages of the methods are discussed with emphasis on the specificity and sensitivity of the histological procedures. Our strategy for detection of TNF mRNA and protein provides a solid basis for clarifying the cellular synthesis, regulation and function of TNF in the normal, injured or diseased CNS. Furthermore, the methodology can readily be applied in studies of other cytokines and growth factors in the CNS.

IFNgamma enhances microglial reactions to hippocampal axonal degeneration.

Glial reactivity is implicated in CNS repair and regenerative responses. Microglia, the cells responding earliest to axonal injury, produce tumor necrosis factor-alpha (TNFalpha), a cytokine with both cytopathic and neuroprotective effects. We have studied activation of hippocampal microglia to produce TNFalpha in response to transection of perforant path axons in SJL/J mice. TNFalpha mRNA was produced in a transient manner, peaking at 2 d and falling again by 5 d after lesioning. This was unlike other markers of glial reactivity, such as Mac-1 upregulation, which were sustained over longer time periods. Message for the immune cytokine interferon-gamma (IFNgamma) was undetectable, and glial reactivity to axonal lesions occurred as normal in IFNgamma-deficient mice. Microglial responses to lesion-induced neuronal injury were markedly enhanced in myelin basic protein promoter-driven transgenic mice, in which IFNgamma was endogenously produced in hippocampus. The kinetics of TNFalpha downregulation 5 d after lesion was not affected by transgenic IFNgamma, indicating that IFNgamma acts as an amplifier and not an inducer of response. These results are discussed in the context of a regenerative role for TNFalpha in the CNS, which is innately regulated and potentiated by IFNgamma.

Microglia and macrophages are the major source of tumor necrosis factor in permanent middle cerebral artery occlusion in mice.

The proinflammatory cytokine tumor necrosis factor (TNF) is known to be expressed in brain ischemia; however, its cellular and temporal appearance is not fully settled. In this study, nonradioactive in situ hybridization for murine TNF mRNA was performed on brain sections from adult C57x129 mice at 6 hours, 12 hours, 24 hours, 2 days, 5 days, or 10 days (six to eight mice per group) after induction of permanent focal cerebral ischemia. Cortical infarct volumes were estimated, and TNF mRNA-expressing cells were counted within the infarct and infarct border using Cast-Grid analysis. At 12 hours, a peak of 19.2 +/- 5.1 TNF mRNA-expressing cells/mm2 was counted, contrasting two to three times lower values at 6 and 24 hours (6.4 +/- 4.6 and 9.2 +/- 3.4 cells/mm2, respectively) and <2 cells/mm2 at 48 hours and later stages. The TNF mRNA-expressing cells were distributed along the entire rostrocaudal axis of the cortical infarcts and occasionally within the caudate putamen. At all time points, TNF mRNA colocalized with Mac-1-positive microglia/macrophages but not with Ly-6G (Gr-1)-positive polymorphonuclear leukocytes. Similarly, combined in situ hybridization for TNF mRNA and immunohistochemistry for glial fibrillary acidic protein at 12 and 24 hours revealed no TNF mRNA-expressing astrocytes at these time points. Translation of TNF mRNA into bioactive protein was demonstrated in the neocortex of C57B1/6 mice subjected to permanent middle cerebral artery occlusion. In summary, this study points to a time-restricted microglial/macrophage production of TNF in focal cerebral ischemia in mice.

Increased synthesis of heparin affin regulatory peptide in the perforant path lesioned mouse hippocampal formation.

Heparin affin regulatory peptide (HARP), also known as pleiotrophin or heparin-binding growth-associated molecule, is a developmentally regulated extracellular matrix protein that induces cell proliferation and promotes neurite outgrowth in vitro as well as pre- and postsynaptic developmental differentiation in vivo. Here we have investigated the expression of HARP mRNA and protein in the perforant path lesioned C57B1/6 mouse hippocampal formation from 1 to 35 days after surgery. This type of lesion induces a dense anterograde and terminal axonal degeneration, activation of glial cells, and reactive axonal sprouting within the perforant path zones of the fascia dentata and hippocampus as well as axotomy-induced retrograde neuronal degeneration in the entorhinal cortex. Analysis of sham- and unoperated control mice showed that HARP mRNA is expressed in neurons and white and gray matter glial cells as well as vascular and pial cells throughout the normal, adult brain. Lesioning induced high levels of HARP mRNA in astroglial-like cells in the denervated zones of fascia dentata and hippocampus as soon as day 2 postlesion. This expression reached maximum at day 4, and declined toward normal at day 7-14. Combined HARP in situ hybridization and glial fibrillary acidic protein (GFAP) immunohistochemical staining and double immunofluorescent stainings for GFAP and HARP at day 4 postlesion showed colocalization of HARP mRNA and protein to hypertrophic GFAP-immunopositive astrocytes in the denervated areas. Finally, the axotomized entorhinal layer II neurons, which expressed high levels of HARP mRNA in the normal brain, exhibited a marked decline in hybridization signal after axotomy. The induction of high levels of HARP mRNA and protein in astrocytes in the denervated areas of fascia dentata and hippocampus is of particular interest as astrocytes and astrocyte-derived factors are known to be implicated in axonal growth and regeneration and in rescuing injured neurons.

Lack of effect of short-term depletion of plasma complement C3 on the survival of syngeneic dopaminergic neurons following grafting into the intact rat striatum.

Metabolically compromised cells may be subject to complement-mediated cytotoxicity. The aim of this study was to clarify to what extent plasma complement C3 might contribute to the low survival (5-20%) of grafted dopaminergic neurons. The survival of intrastriatal cell suspension grafts of syngeneic dopaminergic, tyrosine hydroxylase (TH)-containing neurons was compared in rats subjected to short-term i.v. treatment with 1) cobra venom factor (CVF), or 2) placebo treatment. Depletion of plasma complement C3 by CVF was confirmed by crossed immunoelectrophoresis. With 159 +/- 37 (mean +/- SEM) TH-immunoreactive and 154 + /- 40 TH mRNA-expressing neurons in the CVF-treated rats (n = 9), and 117 +/- 34 TH-immunoreactive and 160 +/- 49 TH mRNA-expressing neurons in placebo rats (n = 6), the CVF treatment did not increase the survival of the grafted dopaminergic neurons. Similarly, CVF had no apparent effect on the astroglial, microglial, or oligodendroglial cell response within and around the graft. The data indicate that depletion of plasma complement C3 at the time of grafting has no effect on the long-term survival of syngeneic ventral mesencephalic dopaminergic neuronal grafts.

muFKBP38: a novel murine immunophilin homolog differentially expressed in Schwannoma cells and central nervous system neurons in vivo.

To better understand the process of multistage carcinogenesis in Schwann cells, we have attempted to isolate novel candidate genes involved in neoplastic progression of mouse malignant Schwannoma cells. The semi-differentiated Schwannoma cell line 56-24 and the less differentiated Schwannoma cell line 64-39 were established from peripheral nerve sheath tumors arising in transgenic mice of the MBP/SV40 large T strain Tg29. By using the chemical cross-linking subtraction technique, we have cloned a novel murine cDNA that detects pronounced expression in 56-24 cells but not in 64-39 cells. The longest open reading frame of the cDNA predicts a peptide showing 95% amino acid sequence homology to the recorded sequence of the human immunophilin homolog huFKBPr38, one of a family of proteins that are thought to interface with a wide range of intracellular signal transduction systems. The predicted open reading frame of the corresponding gene, named muFKBP38, encodes a 38 kDa protein that harbors an FK-binding protein (FKBP) domain that is 36% identical to that of muFKBP52, a three-unit tetratricopeptide repeat and a consensus leucine-zipper repeat. Although muFKBP38 mRNA was detected in both neurons and glial cells, pronounced expression of the immunophilin homolog appeared in various classes of neurons associated with the hippocampal formation, as shown by in situ hybridization analysis of adult mouse brains. Taken together, these data indicate that muFKBP38 is (i) a novel potential marker for semi-differentiated Schwannomas, (ii) may form homomultimers and/or interact with other proteins, and (iii) may have a role in neurons associated with memory function.

Microglia and macrophages are major sources of locally produced transforming growth factor-beta1 after transient middle cerebral artery occlusion in rats.

The potentially neurotrophic cytokine transforming growth factor-beta1 (TGF-beta1) is locally expressed following human stroke and experimental ischemic lesions, but the cellular source(s) and profile of induction have so far not been established in experimental focal cerebral ischemia. This study presents the time course and a cellular localization of TGF-beta1 mRNA, visualized by in situ hybridization combined with immunohistochemical staining for microglia, macrophages, or astrocytes, on brain sections from adult spontaneously hypertensive rats subjected to transient proximal occlusion of their middle cerebral artery. Six hours after ischemia, an early and transient neuronal and microglial expression of TGF-beta1 mRNA was observed in the extraischemic cingulate and frontal cortices. Both early and protracted expression of TGF-beta1 mRNA in the caudate-putamen and neocortical infarcts and in the caudate-putamen penumbra colocalized with OX42/ED1-immunoreactive microglia and macrophages, whereas TGF-beta1 mRNA in the neocortical penumbra colocalized with OX42/ED1-immunoreactive cells of a microglial morphology. No astrocytes were double-labeled. The number of TGF-beta1 mRNA-expressing microglia and macrophages increased strongly during the first week. Thereafter, TGF-beta1 mRNA became increasingly restricted to the neocortical penumbra (3 weeks), and after 3 months it was confined to activated microglia in the anterior commissure. Our data establish activated microglia and macrophages as the major source of TGF-beta1 mRNA following experimental focal cerebral ischemia. Consequently, TGF-beta1-mediated functions may be exerted by microglia both in the early degenerative phase, and later in combination with blood-borne macrophages, in the remodeling and healing phase after focal cerebral ischemia.

Expression of a novel murine phospholipase D homolog coincides with late neuronal development in the forebrain.

Members of the phospholipase D (PLD) superfamily are defined by the conserved HXKXXXXD motif, which is essential for the catalytic function of mammalian PLD. PLD enzymes are thought to play roles in signal transduction and membrane vesicular trafficking in mammalian cells. Here we describe a 54-kDa novel murine polypeptide (designated SAM-9) that is predicted to be a membrane-associated member of the PLD superfamily. SAM-9 shares 40, 30, and 29% amino acid identity with potential orthologs, in vaccinia virus, Caenorhabditis elegans, and Dictyostelium discoideum, respectively, and belongs to a subclass of PLD homologs in which the second HXKXXXXD motif is imperfect and harbors a conserved Asp to Glu substitution. The sam-9 gene has more than eight exons, and the two HXKXXXXD motifs are encoded by two highly conserved exons. The expression of the sam-9 gene is greater in the brain than in non-nervous tissue and appears to be predominantly of neuronal origin. sam-9 expression is pronounced in mature neurons of the forebrain and appears to be turned on at late stages of neurogenesis as revealed by in situ hybridization analysis of sam-9 expression during postnatal development of the hippocampal formation and the primary somatosensory cortex.

Microglial and macrophage reactions mark progressive changes and define the penumbra in the rat neocortex and striatum after transient middle cerebral artery occlusion.

Transient middle cerebral artery occlusion in rats leads to infarction of the lateral part of the striatum and adjacent neocortex, with selective neuronal necrosis in the bordering penumbral zones. Administration of glutamate, cytokine, and leukocyte antagonists have rescued mainly neocortical neurons, indicating differences in the degenerative processes. The aim of this study was, therefore, to describe the microglial/macrophage activation and polymorphonuclear leukocyte recruitment patterns and to correlate these with the ischemia-induced degenerative processes. The analysis showed significant differences in the characteristics and timing of the microglial/macrophage responses between the caudate putamen and neocortical infarct zones, the infarct zones and their associated penumbral zones, as well as between the striatal and the neocortical penumbral zone. Infiltrations with polymorphonuclear leukocytes into the infarct zones were limited and shortlasting and confined to the acutely degenerating striatum and piriform cortex. A delayed, massive infiltration with lipid phagocytes into the caudate putamen infarct markedly contrasted an early recruitment and activation of microglia/macrophages in the adjacent penumbra. Within the neocortex, a later onset of degeneration along the insular-parietal axis was marked by neuronal expression of heat shock protein and a progressive microglial activation with induction of the full repertoire of microglial activation markers, including a widespread microglial major histocompatibility complex (MHC) class II antigen expression. We interpret the present results as delineating two differentially progressing penumbral zones, which are likely to reflect differences in the underlying degenerative processes. Differences in the microglial/macrophage activation pattern attract special attention, as these cells may constitute specific targets for therapeutic intervention.

Reduction of the microglial cell number in rat primary glial cell cultures by exogenous addition of dibutyryl cyclic adenosine monophosphate.

The present work examined the effects induced by dibutyryl cyclic adenosine monophosphate (dB-cAMP) on microglial cells in primary glial cell cultures from newborn rats. Microglial cells were identified by OX42 immunohistochemistry and nucleoside diphosphatase histochemistry. Double staining for astrocytes was carried out by combination with glial fibrillary acidic protein immunolabeling. Addition of 0.25 mM dB-cAMP to the cultures decreased the microglial cell number about sixfold. The findings suggest that the effect of dB-cAMP on the microglial cells might be either a direct action of dB-cAMP on the microglial cells or an indirect effect mediated by the astroglial cells.

Cytokines in cerebral ischemia: expression of transforming growth factor beta-1 (TGF-beta 1) mRNA in the postischemic adult rat hippocampus.

Transient global cerebral ischemia induces selective neuronal degeneration in the adult rat hippocampus, which is both preceded and accompanied by activation of microglia and astrocytes. Altered expression patterns of cytokines and growth factors might influence the postischemic neuron-glial interactions as well as the degenerative neuronal processes. Northern blotting of hippocampal tissue from ischemic animals revealed elevated levels of transforming growth factor beta-1 (TGF-beta 1) mRNA, and in the present in situ hybridization study we examine the endogenous expression and cellular localization of TGF-beta 1 mRNA in the adult rat hippocampus at various intervals following 10 min of global cerebral ischemia. Six hours after ischemia, a diffuse expression of TGF-beta 1 mRNA was found throughout the brain, which further intensified until Day 2 and thereafter subsided. In parallel, a massive increase of signal was observed in the hilus fascia dentata from Day 1 and in area CA1 from Day 2 to 4, both areas displaying selective neuronal degeneration. Peak levels of TGF-beta 1 mRNA were found in the hilus around Day 4, whereas expression in the CA1 area persisted through Day 21, the latest time point examined. A similar biphasic response, consisting of a transient, generalized reaction and a persistent lesion-associated activation in areas undergoing selective neuronal degeneration, was previously described for microglia and is reconfirmed in the present study. Cells of the microglial/macrophage lineage thus include the potent modulatory cytokine TGF-beta 1 in their potential repertoire of responses to both CNS activation and lesioning.

Leukocyte infiltration and glial reactions in xenografts of mouse brain tissue undergoing rejection in the adult rat brain. A light and electron microscopical immunocytochemical study.

Neural mouse xenografts undergoing rejection in the adult recipient rat brain were characterized with regard to infiltrating host leukocytes and reactions of graft and host astro- and microglial cells. Rejection occurred within 35 days with infiltration of the grafts by in particular macrophages and T-cells as well as blood-brain barrier (BBB) leakage for IgG. In the surrounding host brain microglial cells showed increased histochemical staining for nucleoside diphosphatase (NDPase) and increased immunocytochemical expression of complement receptor type 3 (CR3), while astroglial cells displayed an increased immunoreactivity for glial fibrillary acidic protein (GFAP). Light microscopic findings of rat major histocompatibility complex (MHC) antigen class I on microglial cells, endothelial cells and leukocytes were confirmed at the ultrastructural level and extended to include a few astrocytes. Rat and mouse MHC antigen class II was only detected on leukocytes and activated microglia. We suggest that host macrophages and activated host and xenograft microglial cells act in situ as immunostimulatory cells on T-helper cells, and that increased levels of donor MHC antigen class I may further enhance the killer activity exerted by host T-cytotoxic cells.