Bente Finsen

Microglia and macrophages are major sources of locally produced transforming growth factor-β1 after transient middle cerebral artery occlusion in rats

The potentially neurotrophic cytokine transforming growth factor‐β1 (TGF‐β1) 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‐β1 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‐β1 mRNA was observed in the extraischemic cingulate and frontal cortices. Both early and protracted expression of TGF‐β1 mRNA in the caudate‐putamen and neocortical infarcts and in the caudate‐putamen penumbra colocalized with OX42/ ED1‐immunoreactive microglia and macrophages, whereas TGF‐β1 mRNA in the neocortical penumbra colocalized with OX42/ ED1‐immunoreactive cells of a microglial morphology. No astrocytes were double‐labeled. The number of TGF‐β1 mRNA‐expressing microglia and macrophages increased strongly during the first week. Thereafter, TGF‐β1 mRNA became increasingly restricted to the neocortical penumbra (3 weeks), and after 3 months it was confined to activated microglia in the anterior commisure. Our data establish activated microglia and macrophages as the major source of TGF‐β1 mRNA following experimental focal cerebral ischemia. Consequently, TGF‐β1‐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. GLIA 24:437–448, 1998.

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.

Somatostatin and neuropeptide Y in organotypic slice cultures of the rat hippocampus: An immunocytochemical and in situ hybridization study

The neuronal distributions of somatostatin and neuropeptide Y and their respective mRNAs in hippocampal slice cultures were examined by immunohistochemical staining and in situ hybridization. For the in situ hybridization we used an alkaline phosphatase-labelled oligodeoxynucleotide probe for somatostatin mRNA and an 35S-labelled oligodeoxynucleotide probe for neuropeptide Y mRNA. For both neuropeptides the immunostained and hybridized neurons displayed a comparable, organotypic distribution. Most labelled neurons were located in the dentate hilus and stratum oriens of CA3 and CA1. Additional neurons were found in stratum radiatum and pyramidale of CA3, but very few in the corresponding layers of CA1. In all locations the density of somatostatin- and neuropeptide Y-reactive cells exceeded that observed in vivo. Also, the hybridization signal of the individual neurons appeared enhanced in the slice cultures. Methodologically it was noted that the non-radioactive alkaline phosphatase-labelled oligodeoxynucleotide probe gave excellent in situ hybridization results with detailed cellular resolution and no apparent problems of tissue penetration, even when used on whole-mount explants. These results demonstrate that somatostatin and neuropeptide Y-immunoreactive and mRNA containing neurons retain their organotypic distribution and basic morphological characteristics in the slice cultures. The supernormal density of these neurons and their hybridization signals indicate that a transient developmental increase in neuropeptide expression may persist in vitro.

Xenografting of fetal mouse hippocampal tissue to the brain of adult rats: effects of Cyclosporin A treatment

SummaryThis study examines the effect of the immunosuppressive drug Cyclosporin A (CyA) on the survival and differentiation of solid grafts of fetal (E16–17) mouse hippocampi transplanted to the brain of adult rats. The CyA was given as daily subcutaneous injections of 20 mg/kg from the day before transplantation with reduction of the dose to 15 mg/kg after 14 days. Five weeks after transplantation neuron containing xenografts were recovered in 11 out of 17 CyA-treated recipients (65%). After 8 weeks 9 out of 21 grafts were found (43%). In the control groups, treated only with the vehicle olive oil, 8 out of 14 xenografts were recovered after 5 weeks survival (36%) and only 3 out of 17 after 8 weeks (18%). All xenografts were infiltrated with mononuclear lymphocytic-like cells, but the infiltration was least extensive and least dense in the CyA treated animals. An observed correlation between this cellular infiltration and the gliosis in the xenografts suggested that CyA also directly or indirectly influenced the glial reaction. Most surviving xenografts were located next to the lateral ventricles or the choroid fissure. They were organotypically organized with identifyable cell and neuropil layers, and their connectional organization was similar to rat and mouse allografts grafted to adult recipients. In the absence of major extrinsic afferents the intrinsic pathways observed with Timm staining had reorganized according to known principles for aberrant growth and collateral sprouting. Ingrowth of extrinsic host afferents was only demonstrated for AChE positive host fibers. We conclude that CyA treatment of adult rat recipients can increase the survival of intracerebral fetal mouse hippocampal xenografts and reduce the histological signs of rejection. Xenografting combined with CyA treatment thereby permits the use of a wider spectrum of donor neurons for studies of neuronal interaction and repair.

Kindling Induces Transient Changes in Neuronal Expression of Somatostatin, Neuropeptide Y, and Calbindin in Adult Rat Hippocampus and Fascia Dentata

Summary: Fully hippocampus‐kindled rats were examined 1 day and 1 month after the last stimulation for changes in somatostatin (SS)‐, neuropeptide Y (NPY)‐, and calbindin (CaBP)‐immunoreactivity (ir) and SS‐ and NPY‐mRNA in situ hybridization (ISH). One day after the last stimulation, there was marked, bilateral increase in SS‐ and NPY‐ir in the outer part of the dentate molecular layer. The cell bodies of dentate hilar SS‐ and NPY‐containing neurons, known to project to this area, also appeared to display increased immunoreactivity as well as an increased ISH signal for SS and NPY mRNA. Bilateral de novo expression of NPY‐ir in dentate mossy fiber projection to dentate hilus and CA3 was also evident, but we noted no corresponding NPY‐mRNA signal in the parent cell bodies, the dentate granule cells. After 1 month, the levels of NPY‐ir and ISH signal appeared essentially normal. In contrast, the levels of SS apparently were decreased, although not yet normal. CaBP‐ir was markedly and selectively reduced in dentate granule cell bodies, dendrites, and mossy fibers 1 day after the last stimulation, but after I month CaBP‐ir appeared essentially normal. Because kindling, once established, is a permanent phenomenon, the observed transient changes in SS, NPY, and CaBP in specific hippocampal terminal fields and neuronal populations cannot be associated specifically with kindling. Rather, they relate to the repeated high‐frequency stimulations and may serve as protective measures against deleterious effects of such stimulations.

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.

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. Thesam-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 situhybridization analysis of sam-9 expression during postnatal development of the hippocampal formation and the primary somatosensory cortex.

Induction of microglial immunomolecules by anterogradely degenerating mossy fibres in the rat hippocampal formation.

Degeneration of myelinated axonal connections is generally held to provide a strong stimulus for microglial expression of major histocompatibility complex (MHC) class II antigen. The present study demonstrates that strong microglial reactions also are induced by axonal and terminal degeneration of the unmyelinated hippocampal mossy fibres. After destruction of dentate granule cells by focal injections of colchicine (or transection of the mossy fibres) in adult rats, immunocytochemical analysis of the mossy fibre terminal fields in the dentate hilus and regio inferior of hippocampus proper (CA3) revealed profound changes in microglial cells with increased expression of the complement receptor type 3 and induction of MHC class I antigen, leukocyte common antigen, lymphocyte function-associated antigen-1 and MHC class II antigen. The microglial reaction, first detectable 4 days after the lesion, became maximal during the third postlesional week, and had almost vanished 6 weeks after the lesion. From recent studies we know that anterograde degeneration of myelinated Schaffer-collaterals from CA3 to regio superior of hippocampus proper and myelinated entorhinal perforant path fibres to fascia dentata is accompanied by microglial expression of MHC class I antigen, but not class II. Together with the present findings, this demonstrates that myelin debris is neither necessary nor sufficient to induce expression of microglial MHC class II antigen within the hippocampus.

Chapter 14 Immune reactions against intracerebral murine xenografts of fetal hippocampal tissue and cultured cortical astrocytes in the adult rat

Publisher Summary This chapter discusses the immune reactions against the intracerebral murine xenografts of fetal hippocampal tissue and cultured cortical astrocytes in the adult rat. The chapter discusses the effect of immunosuppression with cyclosporin A (CyA) on the cellular infiltration, the expression of major histocompatibilty (MHC) antigens in and around the grafts, and the humoral antibody response in the host rats. Initial observations regarding the possibility that grafted astroglial cells and neurons differ in their ability to elicit host immune responses is presented in the chapter. These observations are from a series of rats grafted with unstimulated murine astrocyte cultures. Xenograft survival can be prolonged by immunosuppression with CyA; but even in grafts with normal histological organization, CyA does not prevent a light infiltration with T-cells. A few of these express the IL-2 receptor found on activated T-cells. These cells may be of importance for long-term graft survival in both normal and immunosuppressed xenograft recipients. The humoral immune response seems not to be correlated with graft survival and appears unaffected by CyA treatment.

Xenografts of mouse hippocampal tissue, exchange of laminar and neuropeptide specific nerve connections with the host rat brain

Immature hippocampal and fascia dentata tissue from embryonic and newborn C57 mice was grafted to the hippocampal region of newborn Kyoto rats. The age of the donor mice varied from embryonic day 13 to the day of birth, and the recipient rats from the day of birth up to 2 days. After survival times of from 5 weeks to 1 year the recipient brains were histologically processed for the tracing of host-xenograft connections by silver staining and electron microscopy of anterograde degeneration, AChE histochemistry, immunohistochemical demonstration of the neuropeptides CCK and enkephalin, and the histochemical Timm sulphide silver method, as well as stained by ordinary cell and fiber stains. The survival of the xenografts depended on the donor age, with less than 10% survival for newborn donors and 60-69% for E13-16 donors. The surviving xenografts developed an organotypic organization and retained a mouse-specific CCK-reactivity in the associational hilodentate system and the dentate mossy fibers. Judged by their positive AChE histochemistry most xenografts received a host rat cholinergic projection when placed in normal cholinoreceptive areas, including areas outside the normal reach of the septo-hippocampal system like the neocortex. Xenografts encroaching on the trajectory of the host rat commissural and perforant path projections or their terminal fields in fascia dentata received laminar and neuropeptide specific host projections. Electron microscopy of host rat perforant path fibers traced to the xenograft dentate molecular layer confirmed the laminar distribution and revealed numerous asymmetric synaptic contacts with spines. An efferent xenograft projection of CCK-reactive mouse mossy fibers into the host CA3 mossy fiber layer demonstrated that this cross-species, mouse to rat innervation also applied to the normal developmental rules, despite the, for the rat abnormal, CCK-content. The formation of laminar and neuropeptide specific mouse-rat nerve connections demonstrates the potentials of intracerebral neuronal grafting in basic and applied neurobiological research by providing new experimental models for the analysis of developmental and functional interactions between nerve cells.