Reactive glia as rivals in regulating neuronal survival

Reactive gliosis is a response noted after nearly every type of CNS injury and involves both activated microglia and astroglia. Although many investigators believe that reactive glia in some way regulate the survival of injured neurons, the influence of glial elements upon damaged neural tissues remains uncertain. To examine relationships between reactive glia and neurons, secretion products from both microglia and astroglia are tested for their effects upon the survival of cultured neurons. Microglia are found to secrete neurotoxic agents, while astroglia are a source of neuronotrophic factors. Similar patterns of soluble factor production are noted for astroglia-rich or microglia-rich regions of rat neocortex damaged by ischemia. These observations suggest that microglia and astroglia compete for control of neuronal survival. Importantly, microglial neurotoxins might hinder the recovery of neurologic function at sites of inflammation.     

Phenotypic diversity and kinetics of proliferating microglia and astrocytes following cortical stab wounds

Brain injury induces reactive gliosis, characterized by increased expression of glial fibrillary acidic protein (GFAP), astrocyte hypertrophy, and hyperplasia of astrocytes and microglia. One hypothesis tested in this study was whether ganglioside GD3+ glial precursor cells would contribute to macroglial proliferation following injury. Adult rats received a cortical stab wound. Proliferating cells were identified by immunostaining for proliferating cell nuclear antigen (PCNA) and by [3H]-thymidine autoradiography, and cell phenotypes by immunocytochemical staining for GD3, GFAP, ED1 (for reactive microglia) and for Bandeiraea Simplicifolia isolectin-B4 binding (all microglia). Animals were labeled with thymidine at 1,2,3, and 4 days postlesion (dpl) and sacrificed at various times thereafter. Proliferating cells of each phenotype were quantified. A dramatic upregulation of GD3 on ramified microglia was seen in the ipsilateral hemisphere by 2 dpl. Proliferating cells consisted of microglia and fewer astrocytes. Microglia proliferated maximally at 2-3 dpl and one third to one half were GD3+. Astrocytes proliferated maximally at 3-4 dpl, and some were also GD3+. Both ramified and ameboid forms of microglia proliferated and by 4 dpl all GD3+ microglia were ED1+ and vice versa. In the contralateral cortex microglia expressed neither GD3 nor ED1. Thus they acquired these antigens when activated. Neither microglia nor astrocytes that were thymidine-labeled at 2, 3, or 4 dpl changed in number in subsequent days. Most thymidine+ astrocytes were large GFAP+ reactive cells that clearly arose from pre-existing astrocytes, not from GD3+ glial precursors. In this model of injury microglia proliferate earlier and to a much greater extent than astrocytes, they can divide when in ramified form, and GD3 is up-regulated in most reactive microglia and in a subset of reactive astrocytes. We also conclude that microglial proliferation precedes proliferation of invading blood-borne macrophages.     

Pregnancy-specific alterations in the expression of the insulin-like growth factor system during early placental development in the ewe

The placenta is recognized as an important determinant of fetal growth rate, yet the factors regulating its proliferation remain poorly understood. Components of the insulin-like growth factor (IGF) system were localized in the ovine uterus using in situ hybridization between days 13-55 of gestation, the period of implantation and placentome formation. IGF-II messenger RNA (mRNA) expression was intense in the fetal mesoderm, particularly at the tips of the invading placentome villi. Moderate levels of IGF-II mRNA were also observed in the maternal caruncular stroma. In contrast, expression of IGF-1 mRNA was low (compared to estrous levels) and ubiquitous decreasing as gestation advanced. IGF-binding protein-2 (IGFBP-2 mRNA was not detected until day 29 of gestation, when it appeared restricted to the dense caruncular-like stroma lining the luminal epithelium, colocalized with IGFBP-4. High concentrations of IGFBP-4 mRNA expression were also found in the placentome capsule. IGFBP-3 mRNA expression was intense in the luminal epithelium between days 13-15 of gestation. Subsequently, levels in this region dropped significantly (P < 0.001). IGFBP-3 mRNA expression was also high in the maternal placentome villi, where photographic emulsions localized expression to blood vessel walls. Peak expression of IGF type 1 receptor (IGF-1R) mRNA was found in the deep uterine glands, with intermediate expression in the superficial uterine glands. Moderate expression of IGF-1R mRNA was initially recorded in caruncular stroma, but levels in this region decreased significantly (P < 0.001) to below the detection limit of the technique after interdigitation by the fetal allantochorion. Furthermore, IGF-1R mRNA could not be detected in any fetal placentome tissue. This study, therefore, has established the pattern of expression of the IGFs, IGF-1R, and three of the IGFBPs during establishment of the ovine placenta. It will form the basis for future work to investigate how this system is regulated and to determine the role of the IGFs in placental development.     

Distinct sites of insulin-like growth factor (IGF)-II expression and localization in lesioned rat brain: possible roles of IGF binding proteins (IGFBPs) in the mediation of IGF-II activity

Although expression of the IGF-II has been demonstrated within the central nervous system (CNS), past studies have failed to reveal its precise roles or responses subsequent to a traumatic injury. To demonstrate that IGF-II, IGFBP, and IGF receptor (-R) expression alters in response to a penetrating CNS injury, we used the techniques of ribonuclease protection assay, in situ hybridization, immunohistochemistry, Western blotting, and RIA. Under normal physiology, IGF-II expression is restricted to the mesenchymal support structures of the brain, including the choroid plexus, where its expression is coincident with that of IGFBP-2. Between 1-7 days post lesion (dpl), in the acute phase following a penetrant wound to the CNS, IGF-II and IGF-IIR protein, but not messenger RNA, were colocalized, with IGF-I, IGF-IR, and IGFBP-1, -2, -3, and -6, to neurons, macrophages, astrocytes, and microglia within the damaged tissue. Within the cerebrospinal fluid (CSF), levels of IGF-II peptide increased to peak at 7 dpl. IGFBP-2, -3, and -6 were also observed within the CSF, with IGFBP-2 predominating and exhibiting an increase in binding efficiency from 7-10 dpl. In the chronic phase of injury (7-14 dpl), an increase in both IGF-II, IGF-IIR and IGFBP-5 messenger RNA and protein was observed specifically and focally in the marginal astrocytes forming the limiting glial membrane of the wound. Thus, our evidence suggests that there are two mechanisms of action for IGF-II within the injured rat brain. During the acute phase, the secretion of IGF-II from the choroid plexus into the CSF is up-regulated, resulting in increased transport of the peptide to the wound. In the CSF, transported IGF-II is complexed to IGFBP-2 and essentially demonstrates an endocrine mode of action with a balance of locally produced IGFBPs modulating its bioactivity in the wound. Later in the wounding response, levels of IGF-II decline in the CSF and the wound neuropil, possibly with the aid of increased IGFBP-5 levels that may help to locally sequester and down-regulate IGF-II activity. Hence, in the chronic phase of the injury response, IGF-II reasserts itself to a predominantly autocrine/paracrine role restricted to the mesenchymal support structures, including the glia limitans, which may help reestablish and maintain tissue homeostasis.     

Astrogliosis in culture: I. The model and the effect of antisense oligonucleotides on glial fibrillary acidic protein synthesis

Astrogliosis is a predictable response of astrocytes to various types of injury caused by physical, chemical, and pathological trauma. It is characterized by hyperplasia, hypertrophy, and an increase in immunodetectable glial fibrillary acidic protein (GFAP). As GFAP accumulation is one of the prominent features of astrogliosis, inhibition or delay in GFAP synthesis in damaged and reactive astrocytes might affect astrogliosis and delay scar formation. The aim of this study is to investigate the possibility of utilizing antisense oligonucleotides in controlling the response of astrocytes after mechanically induced injury. We scratched primary astrocyte cultures prepared from newborn rat cerebral cortex with a plastic pipette tip as an injury model and studied the astrogliotic responses in culture. Injured astrocytes became hyperplastic, hypertrophic, and had an increased GFAP content. These observations demonstrate that injured astrocytes in culture are capable of becoming reactive and exhibit gliotic behaviors in culture without neurons. The increase in GFAP content in injured astrocytes could be inhibited by incubating the scratched culture with commercially available liposome complexed with 3' or 5' antisense oligonucleotides (20 nt) in the coding region of mouse GFAP. The scratch model provides a simple system to examine in more detail the mechanisms involved in triggering glial reactivity and many of the cellular dynamics associated with scar formation. Antisense oligonucleotide treatment could inhibit the GFAP synthesis in injured astrocytes, hence it may be applicable in modifying scar formation in CNS injury in vivo.     

Increased expression of peripheral benzodiazepine receptors in the facial nucleus following motor neuron axotomy

Peripheral benzodiazepine receptors (PBRs) are expressed in a variety of tissues but are normally found at low levels in the brain. Following various types of nerve injury, a reactive gliosis results that exhibits a high expression of this receptor. To further characterize the expression of PBRs following neuronal injury, we evaluated PBR expression in the facial nucleus following facial nerve axotomy (FNA). Injury to a peripheral nerve results in a complex series of metabolic and morphological changes around the injured neuron. Transections of the facial nerve results in a rapid activation of both astrocytes and microglia around axotomized motor neurons. FNA resulted in an increase in the staining for both astrocytes (glial fibrillary acidic protein) and activated microglia (OX42). There was also a reduction in synaptic contacts with the motor nucleus as evidenced by reduced staining for the synaptic marker, synaptophysin. In sections labeled with [3H]-PK11195, the subsequent autoradiograms displayed marked increases in the labeling for PBRs. This increase was observed at 5, 7 and 10 days after nerve transection. The increase was primarily in the level of expression (Bmax), with no change in the affinity of the ligand (Kd). The increase in PBR expression after FNA supports the hypothesis that PBRs can be used as a sensitive marker for CNS injury.     

Neuronal control of MHC class II inducibility in rat astrocytes and microglia

We analysed the inducibility of major histocompatibility complex (MHC) class II molecules of astrocytes and microglia in organotypic hippocampus slice cultures of Lewis rats. Treatment with interferon-gamma (IFN-gamma) resulted in the induction of MHC class II molecules on microglia preferentially in the injured marginal zones of the slice culture, but only sporadically in areas containing intact neuronal architecture. In astrocytes, inducibility of MHC class II molecules was even more strictly controlled. IFN-gamma treatment induced MHC class II expression only in the slice culture zones containing degenerated neurons, and not in the presence of functional neurons. After suppression of spontaneous neuronal activity of the slice culture by the sodium channel blocker tetrodotoxin, MHC class II molecules on astrocytes could be induced by IFN-gamma in areas with intact neuronal architecture, and microglia cells exhibited a higher level of expression. These data suggest that loss of neurons could result in MHC class II inducibility of glial cells, and thus in increased immune reactivity of nervous tissue.     

Differential and time-dependent expression of monocyte chemoattractant protein-1 mRNA by astrocytes and macrophages in rat brain: effects of ischemia and peripheral lipopolysaccharide administration

Increasing evidence indicates a key role of chemoattractant cytokines in the accumulation of leukocytes in the central nervous system (CNS) during the course of inflammatory processes. Monocyte chemoattractant protein (MCP-1/JE), a member of the beta-chemokine (C-C chemokine) family, functions as a potent chemoattractant and activator for monocytes. We have investigated the induction of MCP-1 mRNA using in situ hybridization histochemistry (ISH) and characterized its cellular source by combination of ISH and immunocytochemistry in ischemic rat brains as well as in brains of endotoxin-treated rats. Our results show that 6 h-2 d after middle cerebral artery occlusion (MCAO), MCP-1 mRNA is present in astrocytes surrounding the ischemic tissue (penumbra). At later time points (after 4 d), MCP-1 mRNA is found in macrophages and reactive microglia in the infarcted tissue. Peripheral administration of the bacterial lipopolysaccharide (LPS) induced MCP-1 mRNA throughout the brain in a time-dependent manner (1 h-1 d, peak of expression 6-8 h) and was found in astrocytes. In summary, we have found expression of MCP-1 in (a) astrocytes and to a lesser extent in macrophages/reactive microglia after MCA-occlusion and in (b) astrocytes after peripheral administration of LPS. These findings support that MCP-1 is involved in the CNS response to acute trauma or infection and thus may play a key role in inflammatory processes of the brain.     

Experimental brain injury induces expression of amyloid precursor protein, which may be related to neuronal loss in the hippocampus

Previous reports have demonstrated that some focal brain injuries increase amyloid precursor protein (APP) immunoreactivity in the region surrounding the injury where it was localized, in damaged axons and in pre-alpha 2 cells of the entorhinal cortex. However, to date, APP expression in the hippocampus remote from the impact site has not been comprehensively studied. Therefore, we have evaluated APP expression not only in the locally injured cerebral cortex but also in the hippocampus remote from the impact site. In the present paper, diffuse axonal injury was induced in rats in midline fluid percussion injury. APP expression was examined post injury using Western blot analysis and immunohistochemistry. Western blot analysis demonstrated that the expression of 100-kd APP was increased in both the cerebral cortex and hippocampus 24 h after injury. It then decreased in the hippocampus, but did not change in the cerebral cortex, 7 days after injury. Immunohistochemical studies showed increased immunoreactivity of APP in the neuronal perikarya and reactive astrocytes near the region of injury in the cerebral cortex 24 h to 7 days after injury. In the hippocampus, APP accumulated in the CA3 neurons 24 h and 3 days after injury, although no hemorrhagic lesions were seen at that site. The APP positive neurons in CA3 showed shrunken cell bodies and pyknotic nuclei 3 days after injury, and some of the neurons in CA3 had disappeared by 7 days postinjury. The results of present study suggest that traumatic brain injury induces overexpression and accumulation of APP in neuronal perikarya and that these events are followed by degeneration of CA3 neurons. Further, the decline in APP expression in the hippocampus is thought to be due to neuronal loss in CA3 subsector.     

A protease-resistant form of insulin-like growth factor (IGF) binding protein 4 inhibits IGF-1 actions

Smooth muscle cells (SMC) secrete a serine protease that cleaves insulin-like growth factor (IGF) binding protein (IGFBP)-4 into fragments that have low affinity for IGF-1. When IGFBP-4 is added to monolayer cultures of cell types that do not secrete this protease, IGF-1 stimulation of DNA synthesis is significantly inhibited. In contrast, if cell types that secrete this protease are used, IGFBP-4 is a much less potent inhibitor. These studies were conducted to determine whether proteolysis of IGFBP-4 accounted for its reduced capacity to inhibit IGF-1-stimulated DNA synthesis. The cleavage site in IGFBP-4 that the SMC protease uses was determined to be lysine120, histidine121. A protease-resistant mutant form of IGFBP-4 was prepared, expressed, purified, and tested for biologic activity using porcine SMC cultures. Addition of the protease-resistant mutant resulted in inhibition of DNA and cell migration responses to IGF-1. The inhibition was concentration dependent and was maximal when 500 ng/ml (20 nM) of the mutant was added with 20 ng/ml (2.8 nM) of IGF-1. When the mutant was added in the absence of IGF-1, it had no activity. The results show that cleavage of IGFBP-4 at lysine120, histidine121 results in inactivation of the ability of IGFBP-4 to bind to IGF-1. Creation of a mutant form of IGFBP-4 that was not cleaved by the protease resulted in inhibition of IGF-1-stimulated actions. The results suggest that IGFBP-4 can act as a potent inhibitor of the anabolic effects of IGF-1 and that the variables that regulate protease activity may indirectly regulate IGF-1 actions.     

Up-regulation of insulin-like growth factor binding protein-5 is independent of muscle cell differentiation, sensitive to rapamycin, but insensitive to wortmannin and LY294002

Skeletal myoblast differentiation is stimulated by insulin-like growth factors (IGFs). The autocrine action of IGFs is mediated through the type-1 IGF receptor (IGFR-1) and modulated by IGF binding proteins (IGFBPs) secreted by the cells. The mouse C2 myoblast cell line stably transfected with a vector producing IGF-II antisense RNA was used to show that specific IGFBP expression changes with the state of the cells: high levels of IGFBP-2 messenger RNA (mRNA) were found only in proliferating myoblasts, whereas IGFBP-3 mRNA was induced in quiescent cells. Secretion of IGFBP5 was strongly stimulated during differentiation. Insulin and IGF dose-response experiments showed that up-regulation of IGFBP-5 resulted from IGFR-1 activation. Drugs interfering with IGFR-1 signaling and inhibiting myoblast differentiation had different effects on IGFBP-5 up-regulation. Two phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors, wortmaninn and LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], failed to alter IGFBP-5 up-regulation, which persisted in the absence of differentiation. Rapamycin which indirectly prevents activation of the p70 ribosomal protein-S6 kinase (p70S6k), suppressed IGFBP-5 induction. Because the PI3-kinase inhibitors block p70S6k, neither kinase would be required for IGFR-1-dependent IGFBP-5 induction. In C2 anti-IGF-II myoblasts, IGFBP-5 induction is therefore rapamycin-sensitive and independent of differentiation.     

Intracerebral injection of interferon-gamma inhibits the astrocyte proliferation following the brain injury in the 6-day-old rat

The present study examines the influence of interferon-gamma (IFN-gamma) on the astrocyte proliferation in the rat brain injured within the early period of postnatal development. Six-day-old male rats received a lesion in the left cerebral hemisphere and a single injection of recombinant rat IFN-gamma into the lesion cavity. One or 2 days after the injury the rats were injected with 3H-thymidine. Brain sections were immunostained for glial fibrillary acidic protein (GFAP), subjected to autoradiography, and examined microscopically to record proliferating GFAP-immunopositive astrocytes labeled with 3H-thymidine. In the IFN-gamma-injected rats, a statistically significant decrease in the intensity of reactive astrocyte proliferation was revealed. On day 1 after injury the intensity of astrocyte proliferation showed dose-dependent changes. Relations between the astrocyte reactivity and multiple factors existing in the injured and IFN-gamma-injected brain are discussed. The results represent the first in vivo evidence of a dose-dependent action of IFN-gamma on the astrocyte proliferation in response to injury.     

Rapid estimation of regurgitant volume by the proximal isovelocity surface area method in mitral regurgitation: Can continuous-wave Doppler echocardiography be omitted?

To determine whether heme oxygenase-1 (HO-1) protein is induced by endogenous nitric oxide (NO) in rat glial cultures, we examined the effects of lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), and NO donors such as S-nitroso-N-acetylpenicillamine (SNAP), in mixed glial cells and in vivo rat hippocampus. In cultured glial cells, treatment with LPS induced the expression of 130-kd inducible NO synthase (iNOS) after 6 h, and NO2- accumulation and enhancement of the protein level of 33-kd HO-1 after 12 h. In addition, treatment with SNAP induced HO-1 expression after 6 h. Although NOS inhibitors such as NG-nitro-L-arginine (NNA) and NG-methyl-L-arginine did not change LPS-induced iNOS expression, these inhibitors suppressed both NO2- accumulation and the enhancement of HO-1. Immunocytochemistry showed that treatment with LPS for 24 h induced iNOS immunoreactivity predominantly in ameboid microglia, while this treatment induced HO-1-immunoreactivity in both microglia and astrocytes. In in vivo rat hippocampus, microinjection of LPS plus IFN-gamma, or SNAP after 24 h also induced HO-1 immunoreactivity in reactive microglia and astrocytes. In addition, intraperitoneal administration of NNA inhibited HO-1 immunoreactivity induced by the microinjection of LPS plus IFN-gamma. These results suggest that endogenous NO production by iNOS in microglia causes autocrine and paracrine induction of HO-1 protein in microglia and astrocytes in vitro and in rat brain.     

Biomechanical analysis of limited intercarpal fusion for the treatment of Kienb?ck''s disease: a three-dimensional theoretical study

Using mRNA differential display technique, we have found a differentially expressed band in rat brain, designated HAP2G1, which was the strongest one induced in response to peripheral administration of lipopolysaccharide (LPS). Sequence analysis showed that HAP2G1 cDNA is the rat homologue of the human alpha-chemokine IP-10. Using RT-PCR technique and in situ hybridization, we demonstrate that IP-10 mRNA was expressed only in brain tissue of rats treated with LPS and not in control brain tissue. Using semi-quantitative PCR, we found that both cultured astrocytes and microglia express IP-10 mRNA after treatment with LPS. LPS-induced IP-10 mRNA reached peak levels in rat brain and in cultured microglia at approximately 3 h after treatment with LPS. At 10 h, IP-10 mRNA was markedly decreased, and at 24 h it was low but still detectable by PCR or in situ hybridization. In contrast to unstimulated microglia, unstimulated astrocytes constitutively expressed IP-10 mRNA at a low level. Increased IP-10 expression could possibly be involved in the microglia response to inflammatory stimuli in vivo.     

Acute treatment with corticosteroids decreases IGF-1 and IGF-2 expression in the rat diaphragm and gastrocnemius

Massive doses of methylprednisolone (M) or triamcinolone (T) induced diaphragmatic type IIx/b atrophy, resulting in a leftward shift of the force-frequency curve in rats (). To examine the role of insulin-like growth factors (IGFs) in these changes, IGF mRNA content was measured in costal diaphragm, gastrocnemius, and liver removed from 32 rats treated daily during 5 d either with saline (control, C and pair-fed, PF), M, or T (80 mg/kg). Blood samples were taken to measure IGF-1 serum levels. RNA levels were measured by Northern and dot-blots after hybridization with rat IGF-1 or IGF-2 cDNA probes labeled with alpha-32P. Compared with C (845 +/- 128 ng/ml), IGF-1 serum levels were significantly decreased in M (699 +/- 90 ng/ml, p < 0.001 versus C) and PF animals (505 +/- 33 ng/ml, p < 0.001 versus others) and even more so, in T-treated animals (273 +/- 134 ng/ml, p < 0.001 versus others). Along the same lines, IGF-1 expression in the liver was depressed after corticosteroid treatment and in PF, whereas IGF-2 mRNA content remained unchanged. Compared with C, the relative expression of IGF-1 mRNA in the diaphragm was depressed by 44% and 69% in the M and T groups, respectively (p < 0.0001 versus C), while it was unchanged in PF animals. In the gastrocnemius, IGF-1 expression was reduced after M and T (-51% and -59%, respectively, p < 0.0001 versus C) as well as in PF animals (-40%, p < 0.001 versus C). For IGF-2, a similar pattern of expression was found in the diaphragm and the gastrocnemius. Indeed, IGF-2 mRNA tended to decrease in corticosteroid-treated rats (NS) whereas it was unchanged in PF rats. We conclude that decreased IGF expression after corticosteroid treatment was similar in diaphragm and gastrocnemius and may be responsible for the diaphragmatic changes observed after steroid treatment.     

Recurrent tetany as the first symptom of late manifesting celiac disease]

The insulin-like growth factor (IGF) system appears to be important in the regulation of adrenal growth and hormone synthesis. As IGF-binding proteins (IGFBPs) modify IGF bioactivity, we investigated the expression of IGFBP 1-6 genes in different adrenal tumors and hyperplasias to further clarify the role of the IGF system in adrenal pathophysiology. IGFBP-1 mRNA levels were too low to be detected by Northern blot analysis, but could be found by RT-PCR in some tumors and hyperplastic adrenals. Other IGFBPs were detected by Northern blotting. IGFBP-3 mRNA levels were very low in normal adrenals. In adrenal tumors and hyperplastic adrenals, IGFBP-3 mRNA expression was usually higher than in normal adrenals. In hormonally active adrenocortical carcinomas, IGFBP-2, -4, -5 and -6 mRNA levels were lower than in nonfunctional carcinomas and normal adrenals. The low IGFBP mRNA expression in the hormone-producing carcinomas was associated with high IGF-II mRNA content. In adrenocortical adenomas from patients with Cushing's or Conn's syndrome, mean IGFBP mRNA levels were higher than in normal adrenals or in hormonally inactive adenomas. In nodular and bilateral hyperplasias, IGFBP-2, -3 and -4 mRNA expression was on average higher than in normal adrenals but varied substantially, as did IGFBP mRNA levels in pheochromocytomas. In comparison to normal adrenals, pheochromocytomas expressed on average higher levels of IGFBP-2 and -4 but less IGFBP-5 and -6 mRNAs. Our data show that the six IGFBPs 1-6 are expressed at variable level in adrenal tumors and hyperplasias. The low level of IGFBP mRNAs in hormonally active adrenocortical carcinomas was of particular interest.