Intracerebral transportation and cellular localisation of insulin-like growth factor-1 following central administration to rats with hypoxic-ischemic brain injury.

Insulin-like growth factor-1 (IGF-1) has been shown to be neuroprotective when administered centrally following hypoxic-ischemic (HI) brain injury. However, the cerebral distribution and site of action of IGF-1 after intracerebroventricular (i.c.v.) administration are not known. A unilateral HI brain injury was induced in adult rats by a modified Levine method. Either 3H-IGF-1 alone, or in combination with unlabelled IGF-1, was administered into the lateral ventricle 2 h after injury. The activity of 3H-IGF-1 signal in the potentially injured cortex was compared between two treatment groups using image analysis. The regional distribution and cellular localisation of 3H-IGF-1 were examined autoradiographically in potentially injured hemispheres at 0.5 and 6 h after administration. Tritiated IGF-1 was detected predominantly in the pia mater, perivascular spaces and subcortical white matter tracts 0.5 h after administration and decreased by 6 h (p<0.05). The signals associated with the perivascular spaces and pia mater were not blocked by unlabelled IGF-1, suggesting non-saturable binding in these brain areas. IGF-1 signal was co-localised with IGF binding protein (IGFBP)-2 immunostaining in the white matter tracts where the signal was blocked by unlabelled IGF-1, suggesting competitive association. IGF-1 signal associated with neurons and glia was maximal in the cerebral cortex and less in the CA1-2 subregion of the hippocampus which were blocked by unlabelled IGF-1 (p<0.05). The signals from cortical neurons did not decrease 6 h after administration, suggesting specific and persistent binding to these cells. Our results indicate that centrally administered IGF-1 can be translocated to neurons and glia via the perivascular circulation and the ependymal cell-white matter tract pathways.

Alterations in the neural growth hormone axis following hypoxic-ischemic brain injury.

Recently, there has been considerable interest in determining the role of the growth hormone receptor (GHR) in the central nervous system (CNS). The aim of this study was to investigate the role of circulating growth hormone (GH) and the neural GHR after hypoxic-ischemic (HI) brain injury in the 21-day old rat. We observed growth hormone receptor/binding protein (GHR/BP) immunoreactivity to be rapidly upregulated following a severe unilateral HI injury. There was a biphasic increase with an initial rise occurring in blood vessels within a few hours after injury followed by a secondary rise evident by 3 days post-hypoxia in microglia/macrophages, some of which are destined to express insulin-like growth factor-I (IGF-I). There was also an increased immunoreactivity in reactive astrocytes, some of which were in the process of dividing. Subsequently, we attempted to activate the endothelial GHR/BP which was found to be increased after injury by treating with 15 microgram g-1 day-1 s.c. bGH for 7 days. Circulating concentrations of IGF-I fell after injury and were restored with GH treatment (P=0.001), whereas treatment of normal animals had no effect on serum IGF-I. Peripheral GH treatment increased the cerebrospinal fluid (CSF) concentration of immunoreactive IGF-I in the injured rats (P=0.017). GH treatment also reversed the systemic catabolism caused by the injury but had no significant neuroprotective effects. These results indicate that GH therapy can be used to reverse the systemic catabolism that occurs after CNS injury. In addition, these data suggest a role for the neural GHR during the recovery from brain injury, both in terms of the induction of IGF-I and in terms of glial proliferation.

Neuronal activity induction of the stathmin-like gene RB3 in the rat hippocampus: possible role in neuronal plasticity.

Synaptic activity induces a rapid transcriptional response that is essential for the establishment of long-term neuronal plasticity. Using a differential cloning technique, we have identified a gene induced by seizure activity in the brain as RB3. RB3 is a recently cloned gene belonging to the stathmin family of phosphoproteins. Like SCG10, RB3 is brain-specific, although in situ hybridization results show that the expression of RB3 is more ubiquitous than is that of SCG10. Using genomic DNA sequencing, we show that the 27 amino acid sequence unique to the RB3" transcript is encoded by an alternatively spliced exon, exon 2'. Using a peptide antibody raised against exon 2' to detect RB3" and an anti-Flag antibody to detect an epitope-tagged version of RB3, we show that both proteins are localized to the Golgi apparatus of transfected COS7 cells. Of particular interest, RB3 mRNA, but not SCG10 mRNA, is rapidly induced in the dentate gyrus granule layer of the hippocampus after electrically induced seizure activity as well as stimuli leading to long-term potentiation (LTP). In addition, RB3 mRNA is induced in pheochromocytoma (PC12) cells treated with 250 ng/ml NGF. These results suggest that RB3 may play a role in activity-induced neuronal plasticity and neuronal differentiation.

Co-ordinated and cellular specific induction of the components of the IGF/IGFBP axis in the rat brain following hypoxic-ischemic injury.

Insulin-like growth factor 1 (IGF-1) is induced after hypoxic-ischemic (HI) brain injury, and therapeutic studies suggest that IGF-1 may restrict delayed neuronal and glial cell loss. We have used a well-characterised rat model of HI injury to extend our understanding of the modes of action of the IGF system after injury. The induction of the IGF system by injury was examined by in situ hybridization, immunohistochemistry, Northern blot analysis, RNase protection assay and reverse transcriptase-polymerase chain reaction (RT-PCR). IGF-1 accumulated in blood vessels of the damaged hemisphere within 5 h after a severe injury. By 3 days, IGF-1 mRNA was expressed by reactive microglia in regions of delayed neuronal death, and immunoreactive IGF-1 was associated with these microglia and reactive astrocytes juxtaposed to surviving neurones surrounding the infarct. Total IGF-1 receptor mRNA was unchanged by the injury. IGFBP-2 mRNA was strongly induced in reactive astrocytes throughout the injured hemisphere, and IGFBP-3 and IGFBP-5 mRNA were moderately induced in reactive microglia and neurones of the injured hippocampus, respectively. IGFBP-6 mRNA was induced in the damaged hemisphere by 3 days and increased protein was seen on the choroid plexus, ependyma and reactive glia. In contrast, insulin II was not induced. These results indicate cell type-specific expression for IGF-1, IGFBP-2,3,5 and 6 after injury. Our findings suggest that the IGF-1 produced by microglia after injury is transferred to perineuronal reactive astrocytes expressing IGFBP-2. Thus, modulation of IGF-1 action by IGFBP-2 might represent a key mechanism that restricts neuronal cell loss following HI brain injury.

Insulin-like growth factor-I (IGF-I) immunoreactivity in the Alzheimer's disease temporal cortex and hippocampus.

IGF-I has been shown to enhance neuronal survival and inhibit apoptosis. IGF-I immunoreactivity was examined in the Alzheimer's disease and normal post-mortem human hippocampus and temporal cortex to determine whether IGF-I protein levels are altered in response to neurodegeneration. IGF-I immunoreactivity was induced in a subpopulation of GFAP-immunopositive astroglia in the Alzheimer's disease temporal cortex. These observations raise the possibility that IGF-I has a neuroprotective role in the Alzheimer's disease brain.

The movement of IGF-I into the brain parenchyma after hypoxic-ischaemic injury.

The movement of peptides from CSF into the parenchyma is thought to be slow and diffusion limited. However IGF-I can reduce neuronal loss at distal sites when given centrally 2 h after hypoxic-ischaemic (HI) injury. The present study determined the distribution of [3H]IGF-I given into the lateral ventricle after unilateral HI injury in adult rats. Radioactivity in the injured cortex peaked immediately after administration then rapidly declined. Autoradiography demonstrated radioactivity in the perivascular spaces and in the corpus callosum and external capsule of the injured hemisphere. HPLC and radioimmunoassay confirmed a rise in intracerebral IGF-I levels (from 159 +/- 9 to 401 +/- 88 ng g-1). These data suggest that injury can enhance the movement of IGF-I into the cerebrum via the white matter tracts and perivascular spaces.

In situ evidence for DNA fragmentation in Huntington's disease striatum and Alzheimer's disease temporal lobes.

To test the hypothesis that apoptosis is involved in human brain neurodegenerative disorders, we investigated whether DNA fragmentation occurs in Alzheimer's disease (AD). Huntington's disease (HD) and Parkinson's disease, as well as in temporal lobe epilepsy, using neurologically normal post-mortem human brain tissue as a control. Using in situ end labelling of DNA, we found evidence of DNA fragmentation in cells in temporal cortex and hippocampus from patients with AD and in striatum from those with HD. In contrast, only scattered DNA fragmentation positive cells were detected in the pial surfaces of some of the neurologically normal human brains. Thus, cells in the HD striatum and AD temporal cortex exhibited DNA fragmentation, suggesting that apoptosis may be involved in these disorders.

Mechanisms of delayed cell death following hypoxic-ischemic injury in the immature rat: evidence for apoptosis during selective neuronal loss.

The mechanisms leading to delayed cell death following hypoxic-ischemic injury in the developing brain are unclear. We examined the possible roles of apoptosis and microglial activation in the 21-day-old rat brain following either mild (15 min) or severe (60 min) unilateral hypoxic-ischemic injury. The temporal and spatial patterns of DNA degradation were assessed using gel-electrophoresis and in-situ DNA end-labelling. Microglial activation, mitochondrial failure and cell death were examined using lectin histochemistry, 2,3,5,triphenyl-H-tetrazolium chloride (TTC) staining and acid fuchsin staining, respectively. Selective neuronal death produced by the 15 min injury was associated with the development of apoptotic morphology, DNA laddering and acidophilia from 3 days post-hypoxia. The 60 min injury accelerated this process with some cells showing signs of DNA degradation at 10 h post-hypoxia. However, in the cortex, which developed infarction after the 60 min injury, a different pattern of cell loss occurred. The DNA and mitochondria remained intact, and cells basophilic, until after 10 h post-hypoxia, then widespread necrosis developed by 24 hr. In contrast to regions of selective neuronal loss, DNA degradation was initially random (at 24 hr), with 180bp DNA ladders not detected until 3 days post-hypoxia. There was no morphological evidence of apoptosis. Microglial activation coincided with the onset of DNA degradation in regions of selective neuronal loss but not infarction, suggesting a possible role in selective neuronal death. The results suggest that cortical infarction, which was delayed for at least 10 h, was necrotic, and occurred independently of microglial activation and apoptosis. In contrast, selective neuronal death was apoptotic.

Insulin-like growth factor II is induced during wound repair following hypoxic-ischemic injury in the developing rat brain.

Recent evidence suggests that insulin-like growth factor-I (IGF-I) acts as a neurotrophic factor in the injured CNS. The role of the related peptide IGF-II is unclear. Therefore, we compared the induction of IGF-II in the developing rat brain following mild or severe hypoxic-ischemic (HI) injuries. Ligation of the right carotid artery of 21 day old rats followed by either 15 or 60 min exposure to 8% oxygen led to mild or severe unilateral damage respectively. Brains were collected at 1 day, 3, 5, 7 and 10 days, post-hypoxia. In situ hybridization showed that the 15 min injury (which produced selective neuronal loss) produced no change in basal IGF-II gene expression. However, the 60 min injury, which resulted in cortical infarction and severe neuronal loss in other regions, led to the induction of IGF-II mRNA mainly in the infarcted cortex, from 5-7 days post-hypoxia. Immunohistochemical analysis of brains collected 10 days after the 60 min injury showed that IGF-II immunoreactivity (IR) was also increased, predominantly in damaged regions, but also in the contralateral hippocampus. IGF-II IR was associated with non-neuronal cells that appeared to be microglial-like cells and astrocytes. Together these data suggest that IGF-II may modulate the response of glial cells during recovery from cerebral infarction.

Immediate-early gene protein expression in neurons undergoing delayed death, but not necrosis, following hypoxic-ischaemic injury to the young rat brain.

A unilateral hypoxia-ischaemia (HI) 21-day-old rat preparation was used to assess the effects of HI on the expression of the immediate-early gene proteins (IEGPs) c-Fos/FRAs, Fos B, c-Jun, Jun B, Jun D, Krox 20, Krox 24, and on the mRNA for the neurotrophic factor, brain-derived neurotrophic factor (BDNF). Moderate HI (15 min hypoxia) produced delayed, selective neuronal death and was associated with a rapid induction of c-Fos, Fos B, Jun B, Jun D, and c-Jun proteins, but not Krox 20 protein or BDNF mRNA, in neurons on the side of HI and also a delayed expression of c-Jun (and to a lesser extent c-Fos/FRA's and Fos B) 24-48 h after HI in neurons that underwent delayed neuronal death. Krox 24 showed an initial induction followed by a long-lasting suppression of its expression in regions undergoing cell loss. Severe HI (60 min hypoxia) resulted in seizures and rapid neuronal loss and infarction (necrotic cell death) on the side of HI, and was associated with early induction of c-Fos, Fos B, c-Jun, Jun B, Jun D, Krox 20 and Krox 24 protein and BDNF mRNA in neurons on the non-ligated side of the brain. Fos, c-Jun, Jun B, Jun D and Krox 24, but not Krox 20, Fos B, or BDNF mRNA, were also induced in non-nerve cells on the damaged side of the brain after both moderate and severe HI, and many of these cells appeared to be dividing. Thus, moderate HI induces IEGP's in neurons and non-nerve cells in damaged regions, whereas severe HI induces IEGP's and BDNF in non-damaged regions. c-Jun (and to a lesser extent c-Fos/FRA's) showed a prolonged expression in neurons undergoing delayed, but not necrotic, cell death suggesting that they may be involved in the biochemical cascade that causes selective delayed neuronal death. BDNF was not induced by HI, and therefore, does not appear to play an endogenous neuroprotective role in the CNS.

Brain-derived neurotrophic factor is induced as an immediate early gene following N-methyl-D-aspartate receptor activation.

Recent studies show that focal brain injury, cerebral ischaemia, hypoglycaemia and seizures increase the expression of c-fos and brain-derived neurotrophic factor in brain. Here we report that hippocampal focal brain injury transiently induces the immediate early genes c-fos, jun-B, c-jun and krox-24 (zif-268) messenger RNA and protein and brain-derived neurotrophic factor messenger RNA in rat dentate gyrus neurons, an effect that was blocked by the N-methyl-D-aspartate receptor antagonist MK-801. Prior administration of the protein synthesis inhibitor cycloheximide super-induced immediate early gene messenger RNA, abolished immediate early gene protein induction, but had no effect on injury-mediated induction of brain-derived neurotrophic factor messenger RNA. Thus, while N-methyl-D-aspartate receptor activation results in the induction of both immediate early genes and brain-derived neurotrophic factor messenger RNA, de novo synthesis of immediate early gene proteins is not critical for the increased expression of brain-derived neurotrophic factor messenger RNA seen in brain after focal injury. These results suggest that brain-derived neurotrophic factor is induced after injury as an immediate early gene.

Brain-derived neurotrophic factor expression after long-term potentiation.

Long-term potentiation (LTP) of perforant-path dentate granule cell synapses, in awake rats, was followed by a time-dependent expression of brain-derived neurotrophic factor (BDNF) mRNA in dentate granule cells. This BDNF expression was blocked by the N-methyl-D-aspartate (NMDA) antagonist dizocilpine maleate (MK-801), which also blocked LTP induction, and by sodium pentobarbital, which shortens LTP persistence. These results suggest that BDNF may participate in the NMDA-receptor mediated cascade of events that result in LTP stabilization.

Is c-Jun involved in nerve cell death following status epilepticus and hypoxic-ischaemic brain injury?

Neurons undergoing delayed neuronal death produced by hypoxia-ischaemia (HI) or status epilepticus (SE) showed a massive expression of c-Jun in their nuclei 24 h after the insult. With SE there was also a weaker induction of c-Fos and Jun B in dying neurons. SE induced in the presence of the NMDA antagonist MK-801 produced no delayed c-Jun expression in the hippocampus and nerve cell death did not occur in this region, although there was a delayed c-jun expression in the amygdala/piriform region, and cell death occurred in this area. Activation of central muscarinic receptors with pilocarpine, or block of D2 dopamine receptors with haloperidol, treatments which do not cause neuronal damage, strongly induced Fos and Jun B in hippocampal and striatal neurons, but only induced c-Jun very weakly. Thus, c-Jun may participate in the genetic cascade of events that produce programmed cell death in neurons.

Differential expression of insulin-like growth factor binding proteins (IGFBP) 4 and 5 mRNA in the rat brain after transient hypoxic-ischemic injury.

Recent studies suggest a role for the insulin-like growth factor (IGF) system in the repair of damaged tissue following hypoxic-ischemic injury in the infant rat brain. We have used a unilateral model of hypoxic-ischemic injury to assess the possible involvement of two IGF binding proteins (IGFBPs), IGFBP-4 and IGFBP-5, in the post-asphyxial response. Ligation of the right carotid artery of 21-day-old rats was followed by either 15 min or 60 min exposure to 8% oxygen to produce moderate and severe damage respectively. Using in situ hybridization, the distribution of IGFBP-4 and IGFBP-5 mRNA was determined in brains collected over 10 days following the insult. In the control brains (no damage), both IGFBPs were expressed in distinct regions. IGFBP-4 mRNA was detected in limited areas of the hippocampus and in several cortical layers, while IGFBP-5 mRNA was found primarily in the thalamus. In response to hypoxic-ischemic injury, IGFBP-4 mRNA expression was reduced in regions of neuronal loss, suggesting a neuronal origin for IGFBP-4. The expression of IGFBP-5 mRNA was not altered by the 15 min insult, but was heavily induced from 3 days following the 60 min insult, particularly in the subependymal layer and adjacent white matter on the ligated hemisphere. This suggests that IGFBP-5 may be involved in recovery from severe hypoxic-ischemic injury and may be important in the regeneration of oligodendrocytes.

MK801 induces immediate-early gene proteins and BDNF mRNA in rat cerebrocortical neurones.

Recent studies have shown that MK801, a potent phencyclidine receptor ligand, causes pathomorphological changes in rat cerebrocortical neurones. Here we report that doses of MK801 (1 and 5 mg kg-1) which have been shown to produce pathomorphological changes, induce the expression of immediate-early gene proteins (IEGPs) and brain-derived neurotrophic factor (BDNF) mRNA in rat cerebrocortical neurones. Blockade of central muscarinic receptors which has been shown to prevent MK801-induced pathomorphological changes in cerebrocortical neurones, also prevented MK801-induced expression of IEGPs and BDNF mRNA. The transiently increased expression of BDNF mRNA may be an acute compensatory response of these neurones to MK801-induced injury.