Normal developing rat brain expresses a platelet-derived growth factor B chain (c-sis) mRNA truncated at the 5' end.

The 5' untranslated sequence (5' UTS) of platelet-derived growth factor B (PDGF-B/c-sis) mRNA is highly preserved through evolution, and inhibits translation of downstream coding sequences. In this study, using Northern analysis we identified two PDGF-B/c-sis mRNAs (3.5 kb and 2.6 kb) expressed in normal developing rat brain. In contrast to the constitutive expression of 3.5 kb mRNA, the expression of 2.6 kb mRNA increased markedly in accordance with those stages of brain development at which we had previously demonstrated an increased immunoreactivity for PDGF-B/c-SIS in neurons (Sasahara et al., 1992). By PCR cloning and the RNase protection assay, we determined the complete sequence of rat PDGF-B/c-sis, and found that the 2.6 kb transcript was a form of the 3.5 kb message truncated at the 5' end, and that the predominant 2.6 kb mRNA commenced 15 nt upstream of the signal peptide. Accordingly, it is suggested that the truncation of 5' UTS contributes to the expression of PDGF-B/c-SIS protein in the CNS. Lack of translational inhibitory 5' UTS of PDGF-B/c-sis transcript and resultant efficient protein translation have been reported in only a few transformed cells and cultured umbilical vein endothelial cells. We have extended this knowledge to the developing rat brain, and suggest that a similar mechanism could operate widely in non-transformed tissue in vivo.

Induction of platelet-derived growth factor beta-receptor in focal ischemia of rat brain.

Our previous study on the ischemia-induced expression of platelet-derived growth factor (PDGF)-B chain in the rat brain prompted us to examine expression of PDGF beta-receptor in the ischemic brain. Focal ischemia was induced by permanent tandem occlusion of middle cerebral and common carotid arteries in spontaneously hypertensive rats. Northern analysis revealed that ischemia significantly increased expression of the receptor in the ischemic neocortex at 4 and 7 days (328 +/- 109%; 323 +/- 119%, respectively, over control: n = 4, p < 0.05 versus sham). Neurons in infarct transiently showed increased immunostaining for the receptor at 1 day, whereas neurons in periinfarct area showed sustained and increased immunoreactivity from 1 to 14 days post-ischemia. Reactive glial cells in the external capsule and in molecular layer of the neocortex adjacent to infarct possessed enhanced immunoreactivity from 1 to 21 days. Furthermore, marked immunoreactivity was observed on brain macrophages in infarct and on the abluminal side of capillaries surrounding infarct from 4 to 7 days. These results demonstrated that ischemic insult increases expression of the PDGF beta-receptor at both the mRNA and protein level in the brain, suggesting its important role in cellular cascade of the ischemic brain.

Ischemia induces the expression of the platelet-derived growth factor-B chain in neurons and brain macrophages in vivo.

To elucidate the role of the platelet-derived growth factor (PDGF)-B chain in the brain, we examined its expression in rat brains with focal ischemia. Focal ischemia was induced by permanent tandem occlusion of the middle cerebral and common carotid arteries in spontaneously hypertensive rats (SHRs). Northern analysis demonstrated that ischemia transiently increased mRNA expression of the PDGF-B chain, but not the PDGF-A chain, in the injured neocortex. The larger transcript (3.5 kb) of the B chain gradually increased to threefold by 16 h, whereas the smaller transcript (2.6 kb) of the B chain markedly increased sixfold by 4 h. Immunohistochemistry revealed enhanced immunoreactivity in the neurons in the infarct and in the periinfarct area from 16 h to days 4-7, with a peak at 24 h. Furthermore, the brain macrophages that accumulated in the infarct showed intense immunostaining in their perinuclear region from days 2 to 14, with a peak at days 5-6. The present study demonstrates that ischemia induces the expression of the PDGF-B chain, first in neurons and later in brain macrophages, and suggests an important role of the PDGF-B chain in the healing process of the injured brain.

A mutation in the microtubule-associated protein tau in pallido-nigro-luysian degeneration.

We detected a missense mutation in exon 10 of tau that causes a substitution at codon 279 (N279K) in a Japanese patient with a familial background of parkinsonism and dementia originally described as pallido-nigro-luysian degeneration. This mutation is the same as one seen in a Caucasian family with pallido-ponto-nigral degeneration. The similarities between these two families suggest a common genetic mechanism that may account for the peculiar distribution of neuroglial degeneration with tauopathy.

Acid phosphatase activity in the aorta of spontaneously hypertensive rats and the effect of various antihypertensive drugs.

In an attempt to obtain information about the arterial lysosomal enzymes in hypertension, we biochemically investigated acid phosphatase (Ac-Pase) activity in the aorta of spontaneously hypertensive rats (SHR) and the effects of various antihypertensive drugs. Ac-Pase activity in SHR was always higher than that in age-matched control rats. The enzyme activity tended to increase progressively with advancing age, a tendency which was more pronounced in SHR than in control rats. The aging process expressed by the Ac-Pase activity seems to be accelerated under hypertensive conditions. Antihypertensive drugs such as reserpine, hydrochlorothiazide, hydralazine and propranolol significantly suppressed the rise of blood pressure and decreased the aortic Ac-Pase activity in SHR. In particular reserpine and propranolol lowered Ac-Pase activity more effectively than it did blood pressure. Hypertension as well as catecholamine seem to be involved in the increase in the aortic lysosomal enzyme activity in SHR.

Expression of platelet-derived growth factor B-chain in neointimal smooth muscle cells of balloon injured rabbit femoral arteries.

In order to obtain information about the developmental mechanisms of restenosis after angioplasty, we investigated an association between the expression of platelet-derived growth factors (PDGFs) and neointimal cell accumulation in rabbit femoral arteries subjected to balloon angioplasty. Northern analysis demonstrated that mRNA expression of PDGF B-chain (PDGF-B) increased markedly in the injured arteries, peaking at day 7 (sevenfold), and the transcripts remained augmented until day 21. Also transcripts of PDGF beta-receptor (PDGFR-beta) and alpha-receptor increased by 3- and 2.5-fold, respectively, but those of PDGF A-chain showed only a slight increase (1.5-fold). In situ hybridization and immunohistochemistry demonstrated the concordant expression of mRNA and protein for PDGF-B in the smooth muscle cells (SMCs) of injured vessels throughout the experiment. PDGF-B expression peaked in neointimal SMCs at day 7. In accordance with PDGF-B expression, cellular proliferation in neointima peaked at day 7, being followed by a dramatic increase of neointimal areas thereafter. Further, we demonstrated PDGFR-beta immunoreactivity in these neointimal cells with PDGF-B expression. Our data provide evidence that PDGF-B may stimulate vascular SMC proliferation and contribute to neointimal formation after angioplasty.

Semiquantitative histochemical investigation of lysosomal enzyme activities in the aortic endothelial cells of spontaneously hypertensive rats.

In order to obtain information about changes in lysosomal enzyme activities in the aortic endothelial cells under hypertensive conditions, semiquantitative histochemical investigations of acid phosphatase (Ac-Pase) and N-acetyl-beta-glucosaminidase (NAGase) activities in the aorta of spontaneously hypertensive rats (SHR) were performed on 'Häutchen' monolayer preparations. The aortic endothelial cells in SHR showed increased Ac-Pase and NAGase activities as compared with those in control normotensive rats, and the activities tended to increase with advancing age. The degenerating process of endothelial cells expressed by lysosomal enzyme activity seems to be accelerated by hypertension. The increased lysosomal enzymes may participate in the further development of hypertensive vascular changes.

Effect of hypertension on lysosomal enzyme activities in aortic endothelial cells.

In order to obtain information about the changes in lysosomal enzyme activities in arterial endothelial cells under hypertensive conditions, a biochemical study was performed on 5 lysosomal enzymes, acid phosphatase, N-acetyl-beta-glucosaminidase (NAGase), cathepsin B, cathepsin D and beta-glucuronidase, in endothelial cells isolated by an enzymatic technique from the aorta of spontaneously and renal hypertensive rats, and normotensive control rats. The aortic endothelial cells in the old spontaneously and the renal hypertensive rats showed increased activities of enzymes examined in comparison with those in the age-matched control rats. Endothelial cells in young spontaneously hypertensive rats did not show any elevated enzyme activities compared with those in the controls, and the enzyme activities tended to increase with aging. From this, it is deduced that hypertension activates lysosomal enzyme activities in aortic endothelial cells. The differences in the activities of NAGase, cathepsin B and cathepsin D between hypertensive and control animals increased markedly with advancing age. These activated lysosomal enzymes seem to be involved in the developmental mechanism of arterial endothelial cell injury in hypertension and in further development of hypertensive vascular changes.

Expression of platelet-derived growth factor B-chain and beta-receptor in hypoxic/ischemic encephalopathy of neonatal rats.

Expression of platelet-derived growth factor B-chain and of its specific receptor (beta-receptor) was investigated in immature brains with hypoxic/ischemic injury. After the left common carotid arteries of seven-day-old rats were ligated and pups were placed in a hypoxic chamber, the protein and messenger RNA of both B-chain and beta-receptor were assessed using immunocytochemistry and northern analysis, respectively. Transcripts for B-chain were localized by in situ hybridization. Faint but definite expression of B-chain and beta-receptor was seen in the brains of untreated neonatal controls. Three to 48 h after hypoxia B-chain protein was generally increased above control levels, but focally decreased expression was seen in infarcted areas. Enhanced induction of messenger RNA of B-chain was seen in the both sides of cerebral cortices and hippocampi at 3 h. Strongly increased positivity for B-chain protein and mRNA occurred in the neurons surrounding the infarct. In situ hybridization still showed this up-regulation seven days after hypoxia. Beta-receptor protein expression was enhanced in some neurons immediately surrounding the infarct at 3 h of hypoxia, and marked up-regulation was seen at 16 h. Beta-receptor messenger RNA remained at control levels. Immunocytochemistry showed strong immunoreactivity for the beta-receptor on the neurons surrounding the infarct at 72 h. These results indicate that a neonatal hypoxic/ischemic insult induces neuronal up-regulation of the platelet-derived growth factor B-chain as well as beta-receptor immediately after hypoxia. While this up-regulation is relatively transient in most neurons, sublethal damage to neurons immediately surrounding an infarct induces sustained up-regulation. Through autocrine and paracrine mechanisms, platelet-derived growth factor B-chain molecules may act as a neuroprotective factor in immature brain experiencing with hypoxic/ischemic injury.

Expression of platelet-derived growth factor B-chain in the mature rat brain and pituitary gland.

For better understanding of the role of platelet-derived growth factor (PDGF) B-chain in the brain, the expression of PDGF B-chain was studied in the mature rat brain at both protein and mRNA levels, by assay of PDGF B-chain-related mitogenic activity, Northern blot, in situ hybridization and immunohistochemistry. It was shown that (1) mature rat brain contained substantial PDGF B-chain-related mitogenic activity, (2) significant amounts of two sizes of transcripts (3.5 kb, widely, and 2.6 kb, weakly and in narrower areas) were expressed in the brain, and (3) the transcripts were localized in ubiquitous neurons by in situ hybridization, with the strongest signal in hippocampal pyramidal cells, which distribution almost corresponded with that of the immunoreactive products. The abundant neuronal localization of the transcript and protein, as well as the neuronal expression of the receptor reported elsewhere, suggests the role of the growth factor in neuronal cells as a neuronal regulatory and/or trophic agent acting by autocrine loop or by neuron to neuron interaction. However, there was an apparent discrepancy in part, in the distribution between transcripts and immunoreactivity; that is, transcripts were expressed intensely in the intermediate pituitary lobe with only a scattered immunoreactivity, and the opposite situation was observed in the accessory olfactory nerve and posterior pituitary lobe. This might suggest that PDGF B-chain is transported or secreted in these foci.

A rapid, easy technique for defining detailed anatomical orientation in pre-deparaffinized tissue sections.

During our previous histochemical studies on changes in anterograde and retrograde degeneration following optic nerve transection in rats, it was necessary to make brain sections in which various bilateral components of the visual system were symmetrically included. We discovered a rapid, easy technique that defines a detailed anatomical orientation in pre-deparaffinized tissue sections. The procedure is as follows: several drops of 1% toluidine blue solution are injected with a syringe under the paraffin section on the glass slide after the sections are cut and floated on warm water. After several seconds, the staining solution is removed, and the sections are then ready to be observed under a microscope. The anatomical orientation of the section can easily be defined without deparaffinization. By controlling the condition of sectioning procedures, we could obtain bilateral symmetrical paraffin sections containing all intended components of the visual system. This method is a rapid, simple and reliable way to get tissue sections which contain all of the multiple portions on the same plane.

Decreased immunoreactivity of platelet-derived growth factor B chain-like peptide after axotomy in the dorsal motor nucleus of the vagus nerve.

Platelet-derived growth factor-B chain (PDGF-B) and B chain-specific beta receptor (PDGF-R) were investigated immunohistochemically in the dorsal motor nucleus of the vagus nerve and hypoglossal nucleus after axotomy using antibodies raised against synthetic polypeptides. PDGF-B and PDGF-R immunoreactivity were observed in nerve cell bodies contralateral to the axotomized nerve in both vagal (degenerative) and hypoglossal (regenerative) nuclei. The immunoreactivity for PDGF-B antibody persisted until day 28 after axotomy in the hypoglossal neurons, while that in many neurons in the vagal nucleus diminished after day 3. In the severed vagal nucleus some of the axotomized neurons showed no immunoreactivity for PDGF-B chain, and these changes preceded the decrease in neuronal numbers in the vagal nucleus. The immunoreactivity for PDGF-R antibody showed no marked change in either the vagal or hypoglossal nucleus until day 28 after axotomy. These findings suggest that the decrease in PDGF-B immunoreactivity is not due to a non-specific depletion of cytoplasmic protein in the severed vagal neurons. PDGF, taken up by the nucleus and bound to chromatin, has been reported to exert direct effects on the enhancement of transcription and synthesis of RNA. The decrease in level of PDGF-B chain in the vagal neurons seems to cause the reduction of RNA and protein synthesis, resulting in neuronal degeneration.

Different stability of neurofilaments for trypsin treatment after axotomy in the dorsal motor nucleus of the vagal nerve and the hypoglossal nucleus.

In an attempt to obtain information about changes of neurofilaments in motor neurons after axotomy, we immuno-histochemically investigated the accumulated neurofilaments in the dorsal motor nucleus of the vagal nerve, which shows nerve cell loss and degenerative changes after axotomy, and in the hypoglossal nucleus, which shows regenerative changes. Affected neurons in the hypoglossal nucleus showed intensified immunoreactivities for neurofilament antibodies phosphorylated at the carboxy-terminal, and these reactions disappeared with trypsin treatment. Accumulated neurofilaments in the neuronal perikarya in the dorsal motor nucleus of the vagal nerve and axons in brain stem also showed intensified immunoreactivities for the same antibodies, and these reactions remained positive after trypsin treatment. Anti-ubiquitin antibody preferentially stained accumulated neurofilaments in the affected vagal neurons, while no reaction was found in the affected hypoglossal neurons. Phosphorylated neurofilaments in hypoglossal neurons are vulnerable to trypsin treatment probably because of the blocking of polymerization or the disassembly of neurofilaments due to amino-terminal phosphorylation. In vagal neurons, the deteriorated amino-terminal phosphorylation or hyperphosphorylation at the carboxy-terminal seems to cause the cross-linkage and polymerization of neurofilaments, and densely packed polymerized neurofilaments probably fail in axonal transport resulting in nerve cell degeneration and death in the dorsal motor nucleus.

Specific expression of type II protein kinase c after axotomy in the dorsal motor nucleus of the vagus nerve and the hypoglossal nucleus.

Protein kinase C (PKC) and growth-associated protein-43 (GAP-43) were investigated immunohistochemically in the dorsal motor nucleus of the vagus nerve and the hypoglossal nucleus after axotomy using monoclonal antibodies against type I, II and III PKC and GAP-43. In the control side of both nuclei, anti-type I and II PKC weakly stained neuronal cell bodies, while anti-type III PKC did not show any reaction with neurons. In the axotomized side of both nuclei, anti-type II PKC antibody intensely stained affected nerve cell bodies as well as plasma membrane. Some of the severed neurons showed intensified reactions for both anti-type II PKC and anti-GAP-43 antibodies in the serial sections. These findings suggest that axotomy increases the type II PKC of the severed neurons, and type II PKC seems to phosphorylate some protein, such as GAP-43, and plays some role in the retrograde neuronal reaction.

Increased 5'-nucleotidase activity induced by dibutyryl cyclic AMP treatment of cultured glial cells.

In an attempt to clarify the relationship between ecto-5'-nucleotidase (5'-N) activity and cell differentiation of glial cells, dibutyryl cyclic AMP (dBcAMP), which induces cell differentiation, was administrated to cultured rat glioma cells in logarithmic and confluent phases of cultivation. To evaluate the cellular differentiation, cell morphology and the number of glial fibrillary acidic protein (GFAP) positive cells were examined. Treatment with 1 mM dBcAMP decreased cell proliferation and induced cell differentiation in both the logarithmic and the confluent phases. The number of GFAP-positive cells increased with cellular aging and this tendency was enhanced by dBcAMP administration. Ecto-5'-N activity was higher in dBcAMP treated cells than in non-treated cells in both the logarithmic and the confluent phases. These findings suggest that ecto-5'-N activity of C6 glioma cells is increased by dBcAMP actions and is accompanied by cell differentiation.

Proliferative effects of humoral factors derived from neuroblastoma cells on cultured astrocytes.

The proliferative effects of humoral factors released from N18-RE105 neuroblastoma (NRE) cells on cultured astrocytes were assessed in separate co-culture and conditioned medium studies. In both experimental conditions, the humoral factors derived from neuroblastoma cells had growth-promoting effects on C6 glioma cells of astroglial lineage, but not on primary cultured astrocytes from new-born rat cerebral cortex. It is assumed that neuron-derived humoral factors include astroglial growth factors and that differences in responsiveness between two kinds of cells are probably related to the stages of astroglial maturation processes.

Humoral factors derived from glial cells protect cultured neuroblastoma cells against glutamate toxicity.

The protective effects of glial cells against glutamate cytotoxicity on neuronal cells were studied using clonal neuroblastoma cells and two types of glial cells. Neuronal cells treated with glia-conditioned medium became able to tolerate glutamate toxicity. It is suggested that the preparatory processes against glutamate toxicity might be developed in neurons by the humoral factor(s) released from glial cells.

Hypoxia prevents seizures and neuronal damages of the hippocampus induced by kainic acid in rats.

The effects of hypoxia on the epileptic seizures and neuronal damages induced by kainic acid were studied in rats using hypoxic chamber equipment. Rats treated with kainic acid and placed in atmospheric pressure showed typical limbic seizures and regressive neuronal changes in CA3 and CA4 of the hippocampus, while those kept in a hypoxic chamber with 8.5% O2 and 91.5% N2 showed moderate hypoxia and a slight decline of mean arterial blood pressure. In these hypoxic rats, seizures were completely prevented and there was remarkably less regressive neuronal injury of the hippocampus. Thus hypoxia has a rather ameliorative effect on the occurrence of seizures and excitotoxic neuronal injuries induced by kainic acid. The contribution of oxygen radicals and endogenous adenosine to preventing excitotoxic neuronal damages by kainic acid was discussed.

Platelet-derived growth factor B-chain-like immunoreactivity in injured rat brain.

Platelet-derived growth factor (PDGF) is a potent mitogen for mesenchymal cells and glial cells. For a better understanding of the role of PDGF B-chain in the brain, the expression of PDGF B-chain was examined immunohistochemically in penetrating injury to the rat brain. Shrunken neurons were distributed with enhanced PDGF B-chain-related immunoreactivity (PBRI) in the vicinity of the lesion during a period from day 1 to day 4 post injury. Platelet-derived growth factor B-chain-related immunoreactivity was transiently observed also in the cytoplasm of the numerous brain macrophages in the lesion on day 3 and day 4. These distributions of PBRI in the lesion were closely related to the neovascularization and astrogliosis there. The close time and spatial correlation between the expression of PBRI and cellular responses to injury seen in this study suggests PDGF B-chain has an important role in the healing process of cerebral wound.

Expression of heparin-binding epidermal growth factor-like growth factor in rat brain.

According to a recent report, messenger RNA coding for a member of the epidermal growth factor (EGF) family, heparin-binding EGF-like growth factor (HB-EGF), is expressed in the central nervous system (CNS). To obtain information about the role of HB-EGF in the brain, we carried out Northern analysis, in situ hybridization, and immunohistochemical studies evaluating the distribution and amounts of the growth factor using cDNA HB-EGF probes and an antibody raised against synthetic HB-EGF propeptide. Northern analysis revealed transcripts for HB-EGF in all regions of normal rat brain. Immunohistochemically, HB-EGF was demonstrated extensively in neurons at levels varying according to location. HB-EGF mRNA also was detected in neurons, suggesting that the growth factor is produced in these cells. HB-EGF mRNA and immunoreactivity were also demonstrated in interfascicular oligodendrocytes. These findings suggest that HB-EGF is a physiologic ligand for brain EGF receptors, and is likely to be important in neural function.