Griffonia simplicifolia II lectin labels a population of radial glial cells in the embryonic rat brain.

Staining with the Griffonia simplicifolia II lectin (GSL II), a marker for adult rat oligodendrocytes, was studied in rat brain at embryonic (E) days E14, E16, E18, and E20. At E14, only two regions of the CNS showed labeling--the hippocampal primordium and the ventromedial floor of the mesencephalic aqueduct and fourth ventricle. Labeling in these areas was confined to cells with bipolar processes which spanned from the ventricular lumen to the pial surface. At E16, GSL II+ radial glia were present in the hippocampus, septal area, cerebral peduncle, thalamus, tegmentum, and throughout much of the midbrain. At E16, E18, and E20, GSL II strongly labeled two midline radial glial raphes--dorsal to the mesencephalic aqueduct and ventral to the mesencephalic aqueduct and fourth ventricle. Comparison of GSL II lectin labeling with nestin immunostaining, using Rat-401, showed that GSL II labeled a distinct population of radial cells different from nestin-positive radial glia. GSL II also labeled a group of neurons at E16 and E18 in the hypothalamus and basal striatum. We suggest that GSL II+ radial glia represent a population of oligodendroglial precursors present in the embryonic brain, and that GSL II reactivity could serve as a differentiation marker for cells committed to the oligodendroglial lineage. This work supports the possibility that some oligodendroglia, like astrocytes, are derived from radial glial precursors.

Lectin staining of sheep microglia.

The B4-isolectin from Griffonia simplicifolia is known to stain microglial cells in a variety of species. The present report describes a lectin staining method that has been modified to facilitate staining of resting microglia, as well as perivascular cells, in vibratome sections of normal sheep brain. This modified method employs tissue fixed in formaldehyde or paraformaldehyde and requires incubating sections with Triton X-100 prior to staining.

Cytokine transcripts expressed by microglia in vitro are not expressed by ameboid microglia of the developing rat central nervous system.

Because of morphological similarities between ameboid microglia in the developing central nervous system (CNS), brain macrophages in the injured CNS, and cultured microglia in vitro, it is thought that these cell types are functionally equivalent. To investigate the validity of this assumption, we have compared mRNA levels of interleukin-1alpha and -1beta (IL-1alpha and IL-1beta), tumor necrosis factor-alpha and -beta (TNF-alpha and TNF-beta), transforming growth factor-beta1 (TGF-beta1), and macrophage colony-stimulating factor (M-CSF) in the postnatal day 4 (P4) supraventricular corpus callosum (SVCC) with those in unstimulated cultured microglia. Control tissues included spleen, cortex, hippocampus, and cerebellum. Our analyses have shown that while IL-1alpha, IL-1beta, TNF-alpha, TNF-beta, and TGF-beta1 transcripts are abundantly expressed by cultured microglia, they are very low to virtually undetectable in the SVCC. These data strongly suggest that ameboid microglia, which are concentrated in the SVCC, are unlikely to be a significant source of these cytokines. Our study, which shows clear differences in the functional status of cultured microglia vs. ameboid microglia in vivo, stresses the importance of using caution when interpreting in vitro findings in terms of the in vivo functions of microglia.

Comparative evaluation of cytokine profiles and reactive gliosis supports a critical role for interleukin-6 in neuron-glia signaling during regeneration.

Using reverse transcription polymerase chain reaction (RT-PCR), we have studied the temporal expression of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), transforming growth factor-beta 1 (TGF-beta 1), and tumor necrosis factor-alpha (TNF-alpha) mRNAs in three axotomy paradigms with distinct functional outcomes. Axotomy of adult rat facial motoneurons results in neuronal regeneration, axotomy of neonatal facial motoneurons results in neuronal apoptosis, and axotomy of rubrospinal neurons results in neuronal atrophy. Our RT-PCR findings show that a significant and sustained upregulation of IL-6 mRNA is associated uniquely with the regeneration of adult facial motoneurons. Histochemical studies using IL-6 immunohistochemistry show intense IL-6 immunoreactivity in axotomized adult facial motoneurons. Assessment of reactive glial changes with astroglial and microglial markers reveals that the reactive gliosis following adult facial nerve axotomy is more intense than that observed in either of the other two paradigms. Exposure of cultured microglial cells to IL-6 stimulates microglial proliferation in a dose-dependent manner. Cultured microglia also show expression of IL-6 receptor mRNA, as determined by RT-PCR. Our findings support the idea that reactive gliosis is required for neuron regeneration to occur, and more specifically, they suggest that neuron-derived IL-6 serves as a signalling molecule that induces microglial proliferation during motoneuron regeneration.

Microglial response is poorly correlated with neurodegeneration following chronic, low-dose MPTP administration in monkeys.

Many investigators have reported extensive microglial activation in the mouse substantia nigra and striatum following acute, high-dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration. Our previous work demonstrated tyrosine hydroxylase (TH)-positive fiber sprouting in the striatum in monkeys that had received a partial dopaminergic lesion using a low-dose, chronic MPTP administration paradigm. To characterize the microglial response, we utilized HLA-DR (LN3) to immunolabel the class II major histocompatibility complex (MHC II). In MPTP-treated monkeys, there was an intense microglial response in the substantia nigra, nigrostriatal tract, and in both segments of the globus pallidus. This response was morphologically heterogeneous, with commingled ramified, activated, and multicellular morphologies throughout the extent of these basal ganglia structures. Surprisingly, there was little evidence of microglial reactivity in the striatum despite evidence of neurodegeneration-by silver labeling and by loss of TH immunolabeling. Moreover, this pattern of microglial reactivity was the same in all animals that had received MPTP and seemed to be independent of the degree of neurotoxin-induced neurodegeneration. Thus, we conclude that microglial reactivity, per se, is not consistently associated with neurodegeneration, but depends on regional differences.

Cytokine mRNA profiles in contused spinal cord and axotomized facial nucleus suggest a beneficial role for inflammation and gliosis.

We have studied temporal mRNA expression patterns for interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), macrophage colony stimulating factor (M-CSF), and transforming growth factor-beta1 (TGF-beta1) in two rat injury paradigms with very different cellular inflammatory reactions: contussion of the spinal cord and axotomy of the facial nerve. Our comparative analyses using semiquantitative reverse transcription polymerase chain reaction (RT-PCR) show an early and robust upregulation of IL-1beta, TNF-alpha, IL-6, and M-CSF mRNAs in spinal cord after contusion injury. Peak expression of these mRNAs was transient and returned to control levels by 24 h postinjury. In contrast, expression of IL-1beta and TNF-alpha mRNAs in the axotomized facial nucleus was minimal and delayed, and levels of M-CSF mRNA remained unaltered. Similar to injured spinal cord, the axotomized nucleus showed a dramatic and early upregulation of IL-6 mRNA, but unlike spinal cord, IL-6 mRNA levels subsided only gradually. Both injury paradigms showed gradually increasing levels of TGF-beta1 mRNA which were maximal at 7 days postinjury. RT-PCR analyses were also performed on isolated blood-borne mononuclear cells and neutrophils. The results showed that these cells contain high levels of IL-1beta and M-CSF mRNAs, moderate levels of TGF-beta and TNF-alpha mRNAs, and minimal levels of IL-6 mRNA. The RT-PCR analyses together with histological observations indicate that expression of the proinflammatory cytokines IL-1beta, TNF-alpha, and IL-6 is short-lived and self-limited after contusion injury, and that it occurs primarily within endogenous glial cells. Transient expression of these molecules likely triggers secondary events which may be beneficial to wound repair and regeneration.