Tateo Sakaguchi

Basic fibroblast growth factor prevents thalamic degeneration after cortical infarction

In the focal infarction model of the rat middle cerebral artery (MCA), the thalamus of the occluded side becomes gradually atrophic, mainly because of retrograde degeneration. We determined whether basic fibroblast growth factor (bFGF) administered intracisternally could prevent this thalamic atrophy. We occluded the left MCA through a small cranial opening, and animals were then divided into two groups. One group received intra-cisternal injections of recombinant bFGF (1 μg dissolved in 0.1 ml of saline with 2% rat serum) starting 1 day after occlusion and repeated once a week to a total dose of 4 μg by four injections. The other group received vehicle solution by the same schedule. The animals were perfused and fixed at 28 days after occlusion, and histological examination was made at the level of the caudoputamen and thalamus. In the bFGF-treated rats, the area of the posterior ventral thalamus of the occluded side was 93% of that of the contralateral side, i.e., significantly larger than in the normal saline-treated rats (75%, p < 0.01). The infarction size was not statistically different in the two groups. Microscopic observation indicated that normal-saline-treated animals showed shrinkage and disappearance of thalamic neurons, whereas bFGF-treated groups showed preservation of thalamic neurons. Computerized analysis of the cell size substantiated this observation. To assess the effect of bFGF on astrocytes, bFGF or vehicle solution was injected into normal rats, and their histology was evaluated at 1, 2, and 4 weeks after injection. The bFGF-injected group showed a significant increase in glial fibrillary acidic protein-positive astrocytes in the brain tissue facing the ventriculocisternal system. The results suggest that bFGF prevents retrograde degeneration of thalamic neurons by working directly as a neurotrophic factor of these neurons or by activating astrocytes located in the thalamocortical pathway.

Expression of Growth Inhibitory Factor mRNA After Focal Ischemia in Rat Brain

Growth inhibitory factor (GIF) is a small protein belonging to the metallothionein family that has the capacity to inhibit neuronal survival and neurite formation in vitro. This study was conducted to investigate the role of GIF in the brain afflicted with ischemic injury. We used the in situ hybridization technique and Northern blot analysis to study the changes in GIF messenger RNA (mRNA) expression in a rat focal ischemia model. On the first day, the expression tended to decrease in the hemisphere ipsilateral to the injury. It returned to normal levels on the second day except for the central area of the middle cerebral artery (MCA) territory. On the third and fourth day, the expression increased diffusely in the hemisphere of the affected side, including the subcortical area. Two weeks after ischemia, the GIF mRNA expression increased again but only in the pert-infarcted area. Down-regulation of GIF on the first day in the cortex ipsilateral to the infarction might promote neurite sprouting. The subsequent increase in GIF mRNA expression on the third and fourth day might be a symptom of neurons attempting to inhibit excessive neurite outgrowth, or to protect themselves against toxicity caused by oxygen radicals. The later increase in the limited area around the infarction may be related to astroglial reaction. Growth inhibitory factor may play an important role in regulating the central nervous system after ischemic insults.

RNA synthesis and processing in the gerbil brain after transient hindbrain ischaemia.

Ribonucleic acid (RNA) synthesis was investigated in gerbils subjected to 15 min transient hindbrain ischaemia using [2-14C]uridine autoradiography. Distribution of synthesized RNA in the subcellular fraction of the tissue was detected by differential centrifugation and density gradient separation using Whittakers method. In [2-14C]uridine autoradiography, uptake of the tracer into the RNA fraction was not reduced after transient ischaemia. Distributional analysis of [2-14C]uridine in the subcellular fractions revealed that tracer activity in the P3 (microsomes) fraction decreased in the ischaemic regions and tended to decrease in the P4 (ribosomes) fraction, although not significantly. Tracer activity in the P1 (nuclei and cell debris) and P2 (mitochondria, myelin and nerve ending particles) fractions did not decrease. These results indicate that RNA synthesis in the nuclei is not inhibited by ischaemia, but RNA processing is disturbed by the level of the transport. Modification of RNA synthesis and processing by transient ischaemia may influence protein synthesis.

Messenger RNA and protein expression of basic fibroblast growth factor receptor after cortical ablation

In a previous report we demonstrated that basic fibroblast growth factor (bFGF), as a multipotent neurotrophic factor, could prevent retrograde degeneration of the thalamic neurons after ablation of the somatosensory cortex. To elucidate the mechanism of this bFGF action, we examined changes in FGF receptor (FGFR) mRNA (flg) expression with in situ hybridization. The FGF receptor protein was detected with the immunoblotting method. The FGFR mRNA expression was found to be diffusely increased in the affected cortex. Microscopic observation indicated that FGFR mRNA was expressed in several types of cortical cells including neurons and non-neuronal cells. This increase could be observed as early as 6 hours after surgery and lasted for 48 hours. In the thalamus, however no change in FGFR mRNA signals was observed. Western blotting detected a protein immunoreactive to anti-FGFR antibody. Samples from the periablated cortex showed an increase in FGFR protein. Samples from the thalamus, however, showed no difference in FGFR protein level between the lesion side and the contralateral side. Application of exogenous bFGF in Gelfoam to the cortical ablation cavity did not show any effect on the gene expression or protein level of FGFR. These results suggest that FGFR is diffusely induced throughout the injured cortex in the early phase after injury and that bFGF may play an important role after injury. Topically applied bFGF might thus modulate cellular responses in the cortex and have a neurotrophic effect on the affected thalamic neurons.

Expression of c-fos mRNA after cortical ablation in rat brain is modulated by basic fibroblast growth factor (bFGF) and the NMDA receptor is involved in c-fos expression

Expression of c-fos mRNA after cortical injury was studied using the in situ hybridization technique. Strong signals for c-fos mRNA were observed immediately after cortical ablation in neurons throughout the cortex ipsilateral to the injury. However, this c-fos mRNA expression was transient and disappeared within 6 h after the injury. When basic fibroblast growth factor (bFGF; 1 micrograms) was applied to the site of ablation, c-fos mRNA signals were observed for a much longer period. Even 24 h after injury, diffuse expression of c-fos mRNA was detected throughout the cortex, being mainly confined to non-neuronal cells. Intraperitoneal injection of MK-801 (3 mg/kg), a non-competitive NMDA receptor antagonist, suppressed the expression of c-fos mRNA after cortical ablation. It suppressed both the immediate and late expression induced by cortical ablation and bFGF. The immediate expression of c-fos in neurons is likely to be due to spreading depression, while neuronal-glial interactions would be involved in the mechanism of late c-fos expression by non-neuronal cells. Our results suggest that induction of c-fos after cortical injury can be modulated by topically applied bFGF and that the N-methyl-D-aspartate (NMDA) receptor is involved in c-fos expression not only caused by injury itself but also induced by injury and bFGF. As the immediate early genes regulate secondary gene responses, the induction of c-fos may contribute to neuronal plasticity and bFGF may enhance its effect.

Recombinant basic fibroblast growth factor spares thalamic neurons from retrograde degeneration after ablation of the somatosensory cortex in rats.

Retrograde degeneration of thalamic neurons after cortical ablation has long been recognized. Neuronal loss following axotomy eliminates the possibility of regeneration and might prevent the recovery from axonal injury in patients with brain trauma. We investigated whether CS23, a stable recombinant variant of human basic fibroblast growth factor (bFGF), could protect neurons from retrograde degeneration. Four weeks after ablation of the somatosensory cortex in young female rats, there was extensive neuronal degeneration and loss in the lateral ventro-posterior nucleus (VPL) of the ipsilateral thalamus. When Gelfoam soaked in bFGF(CS23) (1 μg/0.l ml) was applied topically at the time of surgery, this neuronal degeneration in the VPL was markedly reduced and macroscopic atrophy of the lateral and medial ventroposterior nucleus (VPL + VPM) was significantly reduced. In contrast, application of bFGF at three days after surgery failed to prevent retrograde degeneration. These resuts indicate that bFGF can prevent thalamic atrophy after ablation of the somatosensory cortex and that administration of bFGF is only effective in the very early period after brain injury.

Expression of tPA mRNA in the facial nucleus following facial nerve transection in the rat.

PLASMINOGEN activators (PAs) have been suggested to play a role in neuronal migration and glial cell proliferation in the developing CNS. Less is known, however, about the role of PAs in the mature nervous system. To elucidate the role of tissue type plasminogen activator (tPA) in the nervous system we used in situ hybridization to study the expression of tPA mRNA within the rat facial nucleus after facial nerve transection. We also studied the effect of MK-801 on tPA mRNA expression in order to investigate whether the previously reported N-methyl-D-aspartate (NMDA) receptor activation is involved in this model. tPA mRNA was expressed in the ipsilateral facial motoneurones from 6 h after injury. This expression continued for at least 2 weeks after facial nerve transection. Administration of MK-801 before axonal injury did not affect the expression of tPA mRNA in the facial nucleus. These data suggest that tPA might be involved in the regenerative process without NMDA receptor activation in mature facial neurones.

Expression of basic fibroblast growth factor receptor messenger RNA in the periinfarcted brain tissue.

The present study examined whether expression of basic fibroblast growth factor receptor (bFGFR) messenger ribonucleic acid (mRNA) was upregulated by focal ischemia. We have studied the in situ hybridization autoradiography for bFGFR mRNA in the rat model of middle cerebral artery (MCA) occlusion. Male Wistar rats were used for occlusion of the left MCA, and were sacrificed 1, 3, 7 and 14 days after MCA occlusion. In situ hybridization was performed on the brain sections of these animals and sham controls by using 35S-labeled antisense and sense (control) RNA probes for rat bFGFR. Expression of bFGFR mRNA was observed in the periinfarcted area of the rats within 1-14 days after MCA occlusion. Expression was evident in the whole hemisphere of the infarcted side, especially at 1 and 3 days after ischemia, but no expression was detected in the contralateral side. On microautoradiograms, the signals of bFGFR mRNA were detected in both neurons and non-neural cells located in the periinfarcted area. Upregulation of bFGFR mRNA detected in the periinfarcted brain tissue suggests that receptor-mediated action of bFGF may be related to preservation of neurons injured by ischemia.

Blood-Borne Macromolecule Induces FGF Receptor Gene Expression After Focal Ischemia

We have detected fibroblast growth factor receptor (FGFR) gene expression in the focal ischemia model. The FGFR gene expression in neurons can be explained by neuronal network disturbances, but the mechanism of astroglial gene expression remains uncertain. We speculated that blood-borne edema fluid may activate gene expression of astroglias. To prove this hypothesis, we compared the patterns of gene expression of FGFR and distribution of edema fluid by using serial tissue sections of the middle cerebral artery (MCA) ischemia. The left MCA of twenty-four male Wistar rats were occluded, and sacrificed 1, 3, 4, 7 and 14 days later by transcardiac perfusion and fixation. The tissues were sliced thinly to 14 microns sections. Part of the tissue sections was used for in situ hybridization for rat FGFR with [35S]labeled RNA probes. The other part of the sections was used for immunostaining for albumin, immunoglobulin G (IgG) and IgM. The FGFR mRNA expression was evident in the lesion-side hemisphere. In the cortex, neurons mainly expressed FGFR gene in the cortex, whereas astroglias and capillary endothelium expressed FGFR in the corpus callosum and internal capsule. The albumin distributed cortex and white matter of the lesion-side and it extended to the contralateral side. The IgG distributed mainly in the lesion-side white matter, and in part extended to the contralateral side. The IgM only distribute to the infarcted area. When we compared topographical distribution of FGFR in the white matter and pattern of albumin, IgG and IgM distribution, pattern of IgG distribution correlated well to the area of FGFR expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection and partial purification of ischaemia-related neurotrophic activity in the periinfarcted brain tissue

In the rat model of middle cerebral artery (MCA) occlusion, axons originating from the ipsilateral cortical and thalamic neurons are injured by ischaemia. The cortical neurons survive thereafter without retrograde degeneration, but thalamic neurons slowly die because of retrograde degeneration. The fate of these two neurons is remarkably different and may be related to neurotrophic activity induced by ischaemia. We detected ischaemia-related neurotrophic activity, and partially purified the factor. Tissue samples were obtained from the cortex adjacent to the infarction and contralateral corresponding site at 4, 8 and 12 days after occlusion of the MCA. They were homogenated with a culture medium and ultracentrifuged. The supernatant was obtained and used for neurotrophic assay. Foetal cortical neurons were obtained from 17 days rat embryo and cultured. Neurotrophic activity was assayed by applying tissue extract to the culture medium. Application of periischaemic cortical extract obtained at 8 and 12 days after ischaemia improved neuronal survival by 50% and 200% as compared to contralateral cortical extract, respectively. The activity was not detectable at 4 days after ischaemia. The neurotrophic activity disappeared by heating the extract at 90 degrees C for 10 min. We fractionated the extract by saturated ammonium sulphate precipitation, followed by gel-filtered with Superose 12 column. The neurotrophic activity was detected in the precipitation of 30 to 60% saturation fraction of ammonium sulphate. With gel-filtration we separated neurotrophic activity in several fractions, which included marker proteins of 8, 22 and 30 kilodaltons. The activities were only detected in the lesioned side but not in the contralateral side.(ABSTRACT TRUNCATED AT 250 WORDS)