Katrin Trautmann

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 (≥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. J. Comp. Neurol. 487:166–175, 2005.

Heme oxygenase (HO)-1 expressing macrophages/microglial cells accumulate during oligodendroglioma progression.

Heme oxygenase (HO-1, HSP32) catalyzes the oxidation of heme to biliverdin and carbon monoxide, a putative neurotransmitter. In the brain, HO-1 expression has been associated with neuroprotection during oxidative stress and hypoxia. However, consecutive downstream mediation is involved in neoangiogenesis and consequent neoplastic outgrowth. We have analyzed HO-1 expression in 69 oligodendroglioma tissue samples, in rat intracranially transplanted C6 gliomas, and neuropathologically unaltered control brains by immunohistochemistry. Double labeling experiments confirmed the nature of HO-1 expressing cells. Reverse transcription-polymerase chain reaction was used to demonstrate HO-1 gene expression. HO-1 immunoreactivity was predominantly observed in macrophages/microglial cells. The number of HO-1 expressing macrophages/microglial cells was significantly lower in primary oligodendrogliomas than in their matched relapses (P<0.0001) and lower in primary anaplastic oligodendrogliomas than in their relapses (P=0.0006). Prominent accumulation of HO-1 expressing macrophages/microglial cells was observed in perinecrotic areas of both experimental rat and human glioblastoma relapses. HO-1 expressing neurons, macrophages/microglial cells and astrocytes were scattered in areas of infiltrative tumor growth. Surprisingly, HO-1 mRNA was detected in only one glioblastoma multiforme relapse. We conclude from these data that HO-1 expressing macrophages/microglial cells accumulate during oligodendroglioma progression in areas of focal necrosis. However, overall biological function of this phenomenon remains to be determined.

Early infiltration of CD8+ macrophages/microglia to lesions of rat traumatic brain injury

Local inflammatory responses play an important role in mediating secondary tissue damage in traumatic brain injury. Characterization of leukocytic subpopulations contributing to the early infiltration of the damaged tissue might aid in further understanding of lesion development and contribute to definition of cellular targets for selective immunotherapy. In a rat traumatic brain injury model, significant CD8+ cell accumulation was observed 3 days post-injury. The CD8+ cells were strictly distributed to the pannecrotic areas and around the pannecrotic perimeter. The morphology, time course of accumulation and distribution of CD8+ cells were similar to that of reactive ED1+ and endothelial monocyte-activating polypeptide II+ microglia/macrophages, but different from W3/13+ T cells. Further double-labeling experiments confirmed that the major cellular sources of CD8 were reactive macrophages/microglia. Both the location of these CD8+ macrophages/microglia to the border of the pannecrosis and their co-expression of endothelial monocyte-activating polypeptide II and P2X4 receptor suggest they might have a central role in lesion development and might thus be candidates for development of immunotherapeutic, anti-inflammatory strategies.

Cyclooxygenase (COX)-1 expressing macrophages/microglial cells and COX-2 expressing astrocytes accumulate during oligodendroglioma progression.

Cyclooxygenases (COX, prostaglandin endoperoxide synthases, PGG/H synthases) are potent mediators of edema, impeding blood flow and immunomodulation in the pathologically altered brain. Two COX iso-enzymes have been associated with brain disease, the constitutively expressed COX-1 and the cytokine-inducible COX-2. We have used single and double labeling immunohistochemistry to analyse COX-1 and COX-2 expression in twenty-six primary WHO grade II oligodendrogliomas, sixteen primary WHO grade III anaplastic oligodendrogliomas, twenty-seven matched recurrences and ten neuropathologically unaltered brains. COX-1 immunoreactivity was predominantly observed in macrophages/microglial cells. The number of COX-1 expressing macrophages/microglial cells was significantly lower in primary oligodendrogliomas than in primary anaplastic oligodendrogliomas (P<0.0001) and in anaplastic oligodendroglioma relapses (P=0.011). Patients with low COX-1 labeling scores in the primary tumors had significantly longer time to progression and overall survival (P=0.0285) than those with high COX-1 labeling scores. COX-2 immunoreactivity was predominantly observed in disseminated neurons and astrocytes. In glioblastoma multiforme relapses, accumulation of COX-2 expressing astrocytes was observed surrounding areas of focal necrosis. The number of COX-2 expressing astrocytes was significantly (P=0.0471) lower in primary oligodendrogliomas than in high grade oligodendroglioma relapses. These data provide convincing evidence for the differential accumulation of cyclooxygenase isoforms during oligodendroglioma progression in vivo.

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.

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.

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.

Upregulation of frizzled 9 in astrocytomas

Wnt/frizzled (FZD) cascades play important roles in controlling cell fate, proliferation, migration, tissue architecture and organogenesis during embryonic development and in adult organisms. The potential involvement of this pathway in tumorigenesis has been established in several types of cancers. Frizzled 9 (FZD9) is expressed in brain and its aberrant expression in gastric cancer was observed. However, its association with astrocytomas remains unknown therefore we studied FZD9 expression in astrocytomas of different malignancy. In the present study, FZD9 expression in 25 astrocytomas was investigated using immunohistochemistry with specific antibodies. Further FZD9 expression in native human brain tissue and glioblastoma cell line were analysed using real‐time reverse transcription polymerase chain reaction (RT‐PCR). In human astrocytomas, FZD9 immunoreactivity (IR) was observed in both microvessels and neoplastic cells. The percentage of FZD9+ microvessels in relation to FZD9+ vessels was significantly higher in malignant astrocytomas than in low‐grade astrocytomas and positively correlated with the astrocytoma World Health Organization (WHO) grading (r = 1, P = 0.04). Furthermore, the FZD9 IR scores positively correlated with astrocytoma WHO grading (r = 1, P = 0.04) and proliferating activity (r = 0.77, P < 0.001). Real‐time RT‐PCR data showed that FZD9 expression in human glioblastoma was significant higher than in normal brain (P < 0.05) but FZD9 expression was only slightly induced in cobalt chloride‐treated human glioblastoma T98G cells compared with untreated cells (P > 0.05). FZD9 is upregulated in astrocytomas, suggesting that FZD9 could be important in the tumorigenesis of human astrocytomas.

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.

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.