Comparative analysis of annexin-1 in neuroepithelial tumors shows altered expression with the grade of malignancy but is not associated with survival.

In various types of cancer, the expression of members of the annexin family of calcium- and phospholipid-binding anti-inflammatory proteins is dysregulated. Annexin-1 (ANXA1, lipocortin-1) is involved in proliferation, differentiation and apoptosis. It serves as a substrate for the epidermal growth factor receptor (EGFR), which is frequently amplified in primary gliomas. It is unclear how annexin-1 is expressed in various neuroepithelial tumors, and whether there is any association with tumor malignancy or survival. We studied annexin-1 expression in 394 glial neoplasms of all grades of malignancy and 81 normal brain samples by immunohistochemistry using tissue microarrays. The results were validated using western blot and reverse transcription-PCR (RT-PCR). In the normal human brain, the expression of annexin-1 is limited to ependymal cells and subependymal astrocytes, but is also upregulated in reactive astrocytes. Ependymomas and astrocytomas showed significantly higher mean annexin-1 expression levels in the cytoplasm compared with oligodendrogliomas (both: P<0.0001). In addition, nuclear staining of annexin-1 in oligodendroglial tumor cells was significantly reduced (P=0.0002), which may be used as a diagnostic tool for differentiating between astrocytomas and oligodendrogliomas. Although annexin-1 expression in ependymomas decreased with the grade of malignancy, diffuse astrocytomas showed a significant increase in cytoplasmic annexin-1-positive tumor cells. However, survival analysis showed that the expression of annexin-1 is not associated with patient survival. Similar to the EGFR amplification profile, primary glioblastomas had a higher annexin-1 expression level compared with secondary glioblastomas. Thus, annexin-1 upregulation in astrocytomas may contribute to tumor progression and its expression profile is similar to its substrate, EGFR, suggesting a possible regulation thereof.

Immunohistochemical comparative analysis of GFAP, MAP - 2, NOGO - A, OLIG - 2 and WT - 1 expression in WHO 2016 classified neuroepithelial tumours and their prognostic value.

Immunohistochemistry is routinely used in differential diagnosis of tumours of the central nervous system (CNS). The latest 2016 WHO 2016 revision now includes molecular data such as IDH mutation and 1p/19q codeletion thus restructuring glioma classification. Direct comparative information between commonly used immunohistochemical markers for glial tumours GFAP, MAP - 2, NOGO - A, OLIG - 2 and WT - 1 concerning quality and quantity of expression and their relation to the new molecular markers are lacking. We therefore compared the immunohistochemical staining results of all five antibodies in 34 oligodendrogliomas, 106 ependymomas and 423 astrocytic tumours. GFAP expression was reduced in cases with higher WHO grade, oligodendroglial differentiation and in IDH wildtype diffuse astrocytomas. By contrast MAP - 2 expression was significantly increased in diffuse astrocytomas with IDH mutation, while NOGO - A expression was not associated with any molecular marker. WT - 1 expression was significantly decreased in tumours with IDH mutation and ATRX loss. OLIG - 2 was increased in IDH-mutant grade II astrocytomas and in cases with higher proliferation rate. In univariate survival analysis high WT - 1 expression was significantly associated with worse outcome in diffuse astrocytic tumours (log rank p < 0.0001; n = 211; median time: 280 days vs 562 days). None of the markers was prognostic in multivariate survival analysis. Among the evaluated markers MAP - 2, OLIG - 2 and WT - 1 showed the best potential to separate between glioma entities and can be recommended for a standardized immunohistochemical panel.

TLR and NOD2 ligands induce cell proliferation in the rat intact spinal cord.

We demonstrate, by 5-bromo-2'-deoxyuridine (BrdU) tracing, the effects of peripheral administration of toll-like receptor (TLR) and NOD2 ligands (stimulators of the innate immune system) on the proliferation of spinal cord cells. Bolus injection of phosphorothioate oligonucleotides containing CpG motifs had no prominent effects on spinal cord neural progenitor cell proliferation, whereas single intraperitoneal injection of polyinosine-polycytidylic acid (Poly I:C, TLR3 ligand), lipopolysaccharide (LPS, TLR4 ligand), R848 (TLR7/8 ligand), or N-acetylmuramyldipeptide (MDP, Nod2 ligand) temporarily increased the number of BrdU(+) cells in the spinal cord. For Poly I:C- or R848-treated groups, the density of BrdU cells reached maximal levels on days 2 to 3 postinjection and then rapidly declined to baseline levels. Only a few of the proliferating cells were of microglial origin, but BrdU(+)/nestin(+) cells were found, suggestive of a proliferation of local progenitor cells. In addition, stimulation of cell proliferation correlated with activation of the innate immune system, that is, microglial cells. Interestingly, activation and cell proliferation was inhibited by corticosteroid dexamethasone but not by indomethacin. These findings suggest an intricate interaction of phylogenetically ancient cellular processes of the innate immune system and regeneration.

Prolonged lesional expression of RhoA and RhoB following spinal cord injury.

Inhibition of the small GTPase ras homology protein (Rho) or its downstream target, the Rho-associated kinase (ROCK), has been shown to promote axon regeneration and to improve functional recovery following spinal cord injury (SCI) in the adult rat. Here, we have analyzed the expression of RhoA and RhoB following spinal cord injury in order to assess whether Rho is a possible target for late pharmacological intervention. In control spinal cords, RhoA(+) cells were almost absent, whereas RhoB was localized to some ependymal cells, a few microglia, and some dissociated neurons. In injured spinal cords, RhoA(+) and RhoB(+)cells accumulated at perilesional areas and in the developing necrotic core early after injury at day 1. After reaching their maximum levels (RhoA at day 3; RhoB at day 1), RhoA(+) and RhoB(+) cell numbers remained significantly elevated until day 28. In areas remote from the lesion (> or =0.75 mm), a more discrete accumulation of RhoA(+) and RhoB(+) cells was observed, primarily in areas of ongoing Wallerian degeneration. RhoA and RhoB were predominantly expressed by polymorphonuclear granulocytes, ED1(+) microglia/macrophages, oligodendrocytes, some neurons, and swollen axons/neurites. Furthermore, expression was located to lesional, reactive astrocytes and fibroblastoid cells confined to areas of scar formation. Our experiments have determined that most RhoA(+) and RhoB(+) cells (>70%) are of mononuclear origin. The persistent presence of lesional RhoA(+) and RhoB(+) axon/neurite fibers over a period of 4 weeks after injury suggests that Rho inhibition is a putative therapeutic concept also for delayed intervention after SCI.

Expression of P2X4 receptor by lesional activated microglia during formalin-induced inflammatory pain.

P2X4 receptor (P2X4R) is an ion channel gated by adenosine 5'-triphosphate. Here we report the presence and the distribution of P2X4R in rat spinal cord by immunohistochemical analysis in an inflammatory pain model. Peripheral inflammation was induced by subcutaneous injection of 4% formalin into the rat hindpaw. Morphology, spatial localization, and activation state of P2X4R+ cells were described at 1, 5, 7, 14, and 28 days after injury. In normal and saline treated control rats, P2X4R was rarely seen. After formalin administration, an increase of P2X4R+ microglia were observed in the spinal cord dorsal horn on the side ipsilateral to the injection, reaching maximal levels by day 7, and then decreasing to normal levels by day 14. This implicates a role of P2X4R in the spinal inflammatory pain process. Furthermore, formalin-induced region-specific increase in activated microglia was confirmed by ED1 and endothelial monocytes activating polypeptide II (EMAP-II) expression. In conclusion, this is the first demonstration that P2X4R is expressed by microglia in the inflammatory pain.

Microglia activation in rat spinal cord by systemic injection of TLR3 and TLR7/8 agonists.

Here we describe activation of microglia in the rat spinal cord by systemic injections of toll-like receptor agonist polyinosine-polycytidylic acid (poly(I:C), a TLR3 ligand) and R848 (a TLR 7/8 ligand). A significant but transient increase of ED-1+ spinal cord microglia was observed 4 days after a single intraperitoneal (i.p.) injection. Immunostainings by different microglial markers, AIF-1, EMAPII, OX6, P2X(4) receptor (P2X4R), indicated that microglia were not fully activated and tracing of cell proliferation by 5-bromo-2 -deoxyuridine revealed that only a small fraction of proliferating cells were microglia (less than 5%). Thus, these stimulators of the innate immune system have, after peripheral administration, clearly effects on the innate immune system of the spinal cord. This should be considered in the design of clinical trials, as both TLR ligands have been used in patients. As injections of TLR ligands can be used to modulate immune activity in the spinal cord, such agents might be tools to modulate local regenerative processes in the spinal cord.

Experimental autoimmune neuritis induces differential microglia activation in the rat spinal cord.

The reactive spatial and temporal activation pattern of parenchymal spinal cord microglia was analyzed in rat experimental autoimmune neuritis (EAN). We observed a differential activation of spinal cord microglial cells. A significant increase in ED1(+) microglia predominantly located in the dorsal horn grey matter of lumbar and thoracic spinal cord levels was observed on Day 12. As revealed by morphological criteria and by staining with further activation markers [allograft inflammatory factor 1 (AIF-1), EMAPII, OX6, P2X(4)R], reactive microglia did not reach a macrophage-like state of full activation. On Day 12, a significant proliferative response could be observed, affecting all spinal cord areas and including ED1(+) microglial cells and a wide range of putative progenitor cells. Thus, in rat EAN, a reactive localized and distinct microglial activation correlating with a generalized proliferative response could be observed.

Spinal cord glia activation following peripheral polyinosine-polycytidylic acid administration.

Effects of repeated intraperitoneal injections of polyinosine-polycytidylic acid on spinal cord cells were analysed. After each injection, the number of ED-1 microglia significantly increased in rat spinal cords. Expression of endothelial monocyte-activating polypeptide II, however, was not observed. The morphology of microglia indicated an incomplete activation state even after three repeated polyinosine-polycytidylic acid injections. Astrocyte activation was observed after the first injection using glial fibrillary acidic protein staining. Simultaneously with glia activation, hyperalgesia was observed, but the expression of P2X4 receptor, which is considered to be closely associated with hyperalgesia, on microglia was not detected. In sum, our data suggest that repeated peripheral injections of polyinosine-polycytidylic acid might alert the central nervous system through limited activation of microglia and astrocytes.

Expression of P2X4 receptor in rat C6 glioma by tumor-associated macrophages and activated microglia.

Here we report an immunohistochemical analysis of P2X4 receptor (P2X4R), an ATP-gated ion channel, expression in rat C6 glioma model. Striking P2X4R expression was detected in compact and infiltrative tumor growth areas. Expression of P2X4R by perivascular cells was observed in normal control brain and in brain areas not affected by tumor growth. Double-immunolabeling revealed that P2X4R was co-expressed by ED1+, AIF-1+, and EMAP II+ tumor-infiltrating microglia/macrophages; whereas on GFAP+ and nestin+ astrocytes P2X4R was hardly seen. Our results indicate that P2X4R expression defines a distinct subset of tumor-associated macrophages and activated microglia in glioma.

Cyclooxygenase-1 and -2 in brains of patients who died with sporadic Creutzfeldt-Jakob disease.

Cyclooxygenases (COXs) mediate inflammation, immunomodulation, blood flow, apoptosis, and fever in various diseases of the brain. Whereas COX-2 is cytokine inducible, COX-1 is expressed by macrophages/microglial cells that accumulate in pathological foci. We analyzed the localization of COX-1 and COX-2 in postmortem cortex slices of eight patients who died with sporadic Creutzfeldt-Jakob disease (CJD) and four neuropathologically unaltered controls by immunohistochemical double-labeling, reverse transcriptase polymerase chain reaction (RT-PCR), and Western blotting experiments. In healthy brains, COX-1 was expressed by single macrophages/microglial cells and COX-2 by disseminated neurons. In patients with CJD, significantly (p = 0.0195) more COX-1-expressing macrophages/microglial cells were detected adjacent to neurons. COX-2 expression was predominantly observed in neurons, and their number was significantly higher (p < 0.0001) compared to controls. RT-PCR and Western blotting revealed more COX-1 and COX-2 mRNA and protein in one CJD patient than in one control patient. These data show that accumulation of COX-1-expressing macrophages/microglial cells and COX-2-expressing neurons might represent important regulatory mechanisms in the complex process of neuronal degeneration in CJD patients.

Galectin-3 labeling correlates positively in tumor cells and negatively in endothelial cells with malignancy and poor prognosis in oligodendroglioma patients.

BACKGROUND: Galectin-3 modulates cell growth, transformation and metastasis in a wide range of neoplasms. PATIENTS AND METHODS: We analyzed galectin-3 expression in a total of 69 oligodendroglioma tissue samples by immunocytochemistry double labeling and RT-PCR experiments. RESULTS: Galectin-3 expression was observed in oligodendrocytes, endothelial cells and macrophages/microglial cells in areas of solid tumor growth. Significantly fewer galectin-3+ oligodendroglioma cells and macrophages/microglial cells were detected in WHO grade II oligodendrogliomas than in their matched relapses and in WHO grade III anaplastic oligodendrogliomas. Inversely, significantly more galectin-3+ endothelial cells were detected in WHO grade II oligodendrogliomas than in their matched relapses and in WHO grade III anaplastic oligodendrogliomas. Patients with low endothelial galectin-3 labeling scores in primary oligodendrogliomas and anaplastic oligodendrogliomas had significantly shorter time to progression and overall survival than patients with high endothelial galectin-3 labeling scores. CONCLUSION: We conclude from these data that the cell-type specific expression of galectin-3 is differentially involved in oligodendroglioma pathology.

Activated leukocyte cell adhesion molecule is expressed in neuroepithelial neoplasms and decreases with tumor malignancy, matrix metalloproteinase 2 expression, and absence of IDH1R132H mutation.

Diffuse growth of gliomas is based on enhanced cell migration and remodeling of the extracellular matrix. Up-regulation of matrix metalloproteinases in gliomas is associated with a poor prognosis. The activated leukocyte adhesion molecule is considered to be indispensable for conversion of matrix metalloproteinase 2 into its active form. We therefore investigated the expression of activated leukocyte adhesion molecule in 9 malignant glial cell lines, 105 normal/reactive human brain specimens, 248 astrocytomas/glioblastomas, 98 ependymomas, 35 oligodendrogliomas, 10 neurocytomas, 10 primitive neuroectodermal tumors (PNET), and 36 medulloblastomas by immunohistochemistry and in selected cases by reverse transcriptase polymerase chain reaction. Correlation between activated leukocyte adhesion molecule expression and tumor grades and entities, proliferation activity, matrix metalloproteinase 2 expression, prognostic isocitrate dehydrogenase (IDH)1 mutation (R132H) status, O-6-methylguanine DNA-methyltransferase (MGMT) promoter status, or association with patient survival were analyzed. All oligodendrogliomas were strongly activated leukocyte adhesion molecule positive. Numbers of activated leukocyte adhesion molecule positive tumors were higher in glioblastomas (93%) than in diffuse astrocytomas (83%), but mean expression intensity was significantly reduced. Anaplastic ependymomas (68%) exhibited reduced numbers of activated leukocyte adhesion molecule-positive tumors and staining intensity compared with lower-grade ependymomas (85%). Activated leukocyte adhesion molecule expression in gliomas was independent of proliferative activity, MGMT status, patient survival, and age, whereas gliomas with IDH1 (R132H) mutation had significantly higher activated leukocyte adhesion molecule levels than their wild-type counterparts. Matrix metalloproteinase 2-negative glioblastomas exhibited significantly reduced activated leukocyte adhesion molecule expression levels compared with astrocytomas. In summary, our findings indicate that activated leukocyte adhesion molecule expression levels in gliomas are probably linked to other mechanisms than its supposed role as regulator of matrix metalloproteinase 2.

Lesional accumulation of P2X4 receptor+ monocytes following experimental traumatic brain injury.

P2X4 receptor (P2X4R) is an ATP-gated ion channel. ATP is an important messenger in traumatic brain injury. Here, we report expression of P2X4R in rat traumatic brain injury with focus on the early phase, most amenable to therapy. Accumulation of P2X4R+ cells was observed as early as 6 h after injury and continued to increase 4 days post-injury at the lesion and remote areas. Double staining revealed that most P2X4R+ cells co-expressed ED-1, a marker for reactive microglia/macrophages, but not nestin or W3/13. Our data suggest that P2X4R expression defines a subtype of activated microglia/macrophages involved in the early processes following traumatic brain injury.

Lesional RhoA+ cell numbers are suppressed by anti-inflammatory, cyclooxygenase-inhibiting treatment following subacute spinal cord injury.

Inhibition of the small GTPase RhoA or its downstream target Rho-associated coiled kinase (ROCK) has been shown to promote axon regeneration and to improve functional recovery following spinal cord injury (SCI) in the adult rat. RhoA has also been implicated in delayed secondary injury pathophysiology, such as free radical formation and loss of endothelial integrity leading to edema formation. In the present report, we have analyzed the effect of the central nervous system (CNS) permissive, putatively neuroprotective, anti-inflammatory cyclooxygenase-1/-2 (COX-1/-2) inhibitor indomethacin in CNS effective dosage (2 mg/kg/day) on lesional RhoA expression following subacute spinal cord injury. In control rats receiving vehicle alone, RhoA+ cells accumulate at the lesion site (Th8). At day 3 following SCI, the RhoA+ cellular composition is composed prevailingly of microglia/macrophages and polymononuclear granulocytes, but few reactive astrocytes. In contrast, in the verum group, lesional numbers of RhoA cells were reduced by indomethacin treatment by more than 60% (P < 0.0001). Inflammation-dependent RhoA expression accessible by cyclooxygenase inhibition proposes an immune-related mechanism. Our results identify COX blockers as candidates for a safe, synergistic, adjuvant treatment option in combination with cell-specific approaches to Rho inactivation, effectively minimizing the pool of RhoA+ cells at the lesion site following SCI.