Neuroprotective effects of the N-terminal tripeptide of IGF-1, glycine-proline-glutamate, in the immature rat brain after hypoxic-ischemic injury.

Insulin growth factor 1 (IGF-1) has an important role in brain development and is strongly expressed during recovery after a hypoxic-ischemic injury. Some of its central actions could be mediated through the N-terminal tripeptide fragment of IGF-1: Gly-Pro-Glu (GPE). The neuroprotective properties of GPE given after a moderate injury in the developing rat brain were evaluated and the binding sites of [(3)H]GPE characterised by autoradiography. After right unilateral injury, GPE or vehicle (V) was injected in the right lateral ventricle (i.c.v.) or in the peritoneal cavity (i.p.) of 21-day-old rats. The percentage of surviving neurons in CA1-2 of the hippocampus was higher in the animals treated with 30 microg of GPE i.c.v. (V: 7.7+/-4.9%, GPE: 26.4+/-7.5%, P=0.02) and 300 microg i.p. (V: 30.2+/-9.1%, GPE: 68.8+/-10.6%, P=0.02) than in animals receiving vehicle. I.p. injection of 300 microg of GPE (V: 78.4+/-7.5%, GPE: 88.4+/-3.2%, P=0.04) was also neuroprotective in the lateral cortex. I.c.v. injection of [(3)H]GPE suggested binding to glial cells in the white matter tracts, the cortex and striatum as opposed to neurons. Although the precise mode of action of GPE is unknown, this study suggests that local administration of GPE is neuroprotective after brain HI injury via glial cells. In addition, systemic administration of GPE showed a more widespread neuroprotective effect. GPE may represent a complementary pathway for central and systemic IGF-1's antiapoptotic effects.

Growth hormone as a neuronal rescue factor during recovery from CNS injury.

There is growing evidence to suggest that growth hormone plays a role in the growth and development of the CNS. Specifically, growth hormone has been implicated in promoting brain growth, myelination, neuronal arborisation, glial differentiation and cognitive function. Here we investigate if growth hormone has a role in the recovery from an unilateral hypoxic-ischaemic brain injury. Using moderate (15 min hypoxia) and severe (60 min hypoxia) models of hypoxic-ischaemia in juvenile rats and standard immunohistochemical techniques, we found intense growth hormone-like immunoreactivity present within regions of cell loss by 3 days (P<0.05). Growth hormone-like immunoreactivity was observed on injured neurones, myelinated axons, glial cells within and surrounding infarcted tissue and on the choroid plexus plus ependymal cells within the injured hemisphere. The pattern of immunoreactivity suggests that (a) growth hormone (or a growth hormone-like substance) is transported via the cerebrospinal fluid and (b) that growth hormone (or a growth hormone-like substance) is acting in a neurotrophic manner specifically targeted to injured neurones and glia. To test this hypothesis we treated a moderate hypoxic-ischaemic brain injury with 20 microg of rat growth hormone by intracerebroventricular infusion starting 2 h after injury (n=12/group). After 3 days the animals were killed and the extent of neuronal loss quantified. Growth hormone treatment reduced neuronal loss in the frontoparietal cortex (P<0.001), hippocampus (P<0.01) and dorsolateral thalamus (P<0.01) but not in the striatum. This spatial distribution of the neuroprotection conveyed by growth hormone correlates with the spatial distribution of the constitutive neural growth hormone receptor, but not with the neuroprotection offered by insulin-like growth factor-I treatment in this model. These results suggest that some of the neuroprotective effects of growth hormone are mediated directly through the growth hormone receptor and do not involve insulin-like growth factor-I induction.In summary, we have found that a growth hormone-like factor increased in the brain in the days after injury. In addition, treatment with growth hormone soon after an hypoxic-ischaemic injury reduced the extent of neuronal loss. These results further suggest that a neural growth hormone axis is activated during recovery from injury and that this may act to restrict the extent of neuronal death.

The window of opportunity for neuronal rescue with insulin-like growth factor-1 after hypoxia-ischemia in rats is critically modulated by cerebral temperature during recovery.

Insulin-like growth factor (IGF-1) is induced in damaged brain tissue after hypoxia-ischemia, and exogenous administration of IGF-1 shortly after injury has been shown to be neuroprotective. However, it is unknown whether treatment with IGF-1 delayed by more than a few hours after injury may be protective. Hypothermia after brain injury has been reported to delay the development of ischemic neuronal death. The authors therefore hypothesize that a reduction in the environmental temperature during recovery from hypoxia-ischemia could prolong the window of opportunity for IGF-1 treatment. Unilateral brain damage was induced in adult rats using a modified Levine model of right carotid artery ligation followed by brief hypoxia (6% O2 for 10 minutes). The rats were maintained in either a warm (31 degrees C) or cool (23 degrees C) environment for the first 2 hours after hypoxia. All rats were subsequently transferred to the 23 degrees C environment until the end of the experiment. A single dose of IGF-1 (50 microg) or its vehicle was given intracerebroventricularly at either 2 or 6 hours after hypoxia. Histologic outcome in the lateral cortex was quantified 5 days after hypoxia. Finally, cortical temperature was recorded from 1 hour before and 2 hours after hypoxia in separate groups of rats exposed to the "warm" and "cool" protocols. In rats exposed to the warm recovery environment, IGF-1 reduced cortical damage (P < 0.05) when given 2 hours but not 6 hours after insult. In contrast, with early recovery in the cool environment, a significant protective effect of IGF-1 in the lateral cortex (P < 0.05) was found with administration 6 hours after insult. In conclusion, a reduction in cerebral temperature during the early recovery phase after severe hypoxia-ischemia did not significantly reduce the severity of injury after 5 days' recovery; however, it markedly shifted and extended the window of opportunity for delayed treatment with IGF-1.

Melanocortin-4 receptor messenger RNA expression is up-regulated in the non-damaged striatum following unilateral hypoxic-ischaemic brain injury.

Melanocortin peptides (alpha-melanocyte-stimulating hormone, adrenocorticotropin and fragments thereof) have been shown to have numerous effects on the central nervous system, including recovery from nerve injury and retention of learned behaviour, but the mechanism of action of these peptides is unknown. A family of five melanocortin receptors have recently been discovered, two of which (melanocortin-3 and melanocortin-4 receptors) have been mapped in the rat brain. We have tested the hypothesis that the expression of one or more of the messenger RNAs for three melanocortin receptors (melanocortin-3, melanocortin-4 and melanocortin-5 receptors) would be altered in rat brain following unilateral transient hypoxic-ischaemic brain injury. In this study, using in situ hybridization, we show that melanocortin-4 receptor messenger RNA was up-regulated in the striatum in the non-damaged hemisphere within 24 h after severe hypoxic-ischaemic injury compared with control brains (P<0.05). In a small group of animals, this induction was not blocked by treatment with the anticonvulsant, carbamazepine. Expression of melanocortin-3 receptor messenger RNA in the brain was not altered in this hypoxic-ischaemic injury model and melanocortin-5 receptor messenger RNA was not detected in either control or hypoxic-ischaemic injured rat brains. We hypothesize that the up-regulation of melanocortin-4 receptor messenger RNA expression in the contralateral striatum may be involved in transfer of function to the uninjured hemisphere following unilateral brain injury.

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.

Bax expression in mammalian neurons undergoing apoptosis, and in Alzheimer's disease hippocampus.

Recent studies indicate that the proto-oncogene Bax, and other related proteins (eg Bcl-2) may play a major role in determining whether cells will undergo apoptosis under conditions which promote cell death. Increased expression of Bax has been found to promote apoptosis, while over-expression of Bcl-2 can inhibit apoptosis. To investigate the role of Bax in nerve cell death in the rat brain we examined the level of Bax expression in cells undergoing apoptosis, using a hypoxic-ischemic stroke model. We found that Bax was expressed at high levels in the nuclei of neurons in the hippocampus, cortex, cerebellum, and striatum on the control side, and that Bax levels increased in hippocampal neurons undergoing apoptosis on the stroke side, and then declined (correlating with cell loss). In the Alzheimer's disease hippocampi we found a concentrated localisation of Bax in senile plaques, which correlated with the localisation of beta-amyloid protein in adjacent sections from the same brains. beta-Amyloid positive plaques are thought to contribute to the Alzheimer's disease process, possibly via an apoptotic mechanism, and this may occur via an increase in Bax in these areas. Bax was also strongly stained in tau-positive tangles in Alzheimer's disease hippocampi, suggesting Bax may play a role in tangle formation. In addition, we observed a loss of Bax expression in the dentate granule cells of Alzheimer's disease hippocampi compared with moderate Bax expression in control hippocampi, and this loss may be related to the survival of these neurons in Alzheimer's disease. Finally, we observed substantially different staining patterns of Bax using three different commercially available antisera to Bax, indicating the need for caution when interpreting results in this area.

The effect of prolonged modification of cerebral temperature on outcome after hypoxic-ischemic brain injury in the infant rat.

Hypoxic-ischemic injuries can evolve over several days, and recent studies suggest that further neuronal death may occur 6 to 72 h later. Because cerebral temperature is an important determinant of outcome during the primary injury, we investigated the effect of temperature, on outcome, during the later phases of injury. Hypoxic-ischemic injury was induced in 21-d-old rats by unilateral ligation of the right carotid artery followed by exposure to 15 min of hypoxia of 8% O2 at 34 degrees C. Cerebral temperature changes were induced by modifying environmental temperature. The rats were divided into four treatment groups: group 1 (n = 15) remained at 34 degrees C for 72 h; group 2 (n = 14) were kept at 34 degrees C for 6 h and then at 22 degrees C for the remaining 66 h; group 3 (n = 17) remained at 22 degrees C for 6 h and 34 degrees C for the next 66 h; group 4 (n = 16) remained at 22 degrees C for 72 h. Rats kept at 22 or 34 degrees C had cortical temperatures of 35.5 +/- 0.1 degrees C and 37.9 +/- 0.2 degrees C, respectively. Histologic outcome was assessed 72 h after hypoxia. The area of cortical infarction was reduced in group 4 compared with groups 1-3 (p < or = 0.05). Striatal damage was reduced in group 4 (p = 0.05). Hippocampal neuronal loss was not significantly altered. In a subsequent study the area of cortical infarction was 12.1 +/- 3 mm2 in group 1 (n = 11) compared with 3.4 +/- 1.5 mm2 group 4 treated rats (n = 10) 21 d after the injury (p < 0.01). Thus hypothermia spanning both the first 6 h and from 6 to 72 h after injury was needed to improve outcome. Conversely exposure to the thermoneutral environment exacerbated the injury. These observations suggest that prolonged moderate cerebral hypothermia can be used to suppress the cytotoxic processes that occur after hypoxic-ischemic injury.

Delayed vasodilation and altered oxygenation after cerebral ischemia in fetal sheep.

The study investigated the hypothesis that delayed cerebral injury after transient cerebral ischemia is associated with vasoconstriction and decreased cerebral oxygenation. Eight chronically instrumented, late gestation fetal sheep were subjected to 30 min of cerebral ischemia in utero. Cortical impedance (CI) and electrocorticogram (ECoG) were recorded to determine the time course of cellular dysfunction. Histologic outcome was assessed 4 d postischemia. Changes in cerebral vascular tone and oxygenation were observed during and for 4 d after the insult using near infrared spectroscopy to measure changes in total cerebral Hb ([tHb]), oxyhemoglobin ([Hbo2]), and oxidized cytochrome aa3 ([Cyto2]). Results are expressed as mean +/- SEM. CI increased transiently during ischemia; then a delayed increase commenced 17.5 +/- 2.3 h postischemia and peaked at 42.3 +/- 2.4 h. ECoG was depressed during and after the insult. Seizures started 13.6 +/- 3.0 h postinsult and persisted for 25.4 +/- 3.2 h. Increases in [tHb] indicated two periods of cerebral vasodilation: immediately after early reperfusion, lasting 2.3 +/- 0.4 h and peaking to 20 +/- 2.0 mumol.L-1; and a later phase, commencing 12.8 +/- 2.0 h postischemia, peaking to 43 +/- 4.0 mumol.L-1 and lasting 43.1 +/- 5.2 h. [Hbo2] was relatively elevated (18 +/- 3.0 mumol.L-1) during d 4 postischemia, demonstrating a delayed increase in mean cerebral oxygen saturation. [Cyto2] fell during the insult (-0.7 +/- 0.2 mumol.L-1); and, commencing at 28-30 h postischemia, fell progressively to reach a minimum of -5.0 +/- 2.8 mumol.L-1 at 78-80 h postischemia. A greater fall in [Cyto2] was related to worse cerebral injury (p < 0.05). Delayed cerebral injury is accompanied by vasodilation and increased mean cerebral oxygen saturation, although a progressive fall in [Cyto2] might indicate a fall in mitochondrial oxygenation, cell loss, or changes in tissue optical characteristics.

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.

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.

Two models for determining the mechanisms of damage and repair after hypoxic-ischaemic injury in the developing rat brain.

Hypoxic-ischaemic (HI) brain injury can lead to selective neuronal loss or pannecrosis. Different mechanisms of damage and recovery may be associated with either pattern of cell loss. Two preparations were developed to investigate the role of growth factors and other mechanisms associated with either of these patterns of damage in the developing brain. Twenty-one-day-old Wistar rats underwent permanent unilateral right carotid artery ligation. Following surgery, ligated rats were warmed for 2 h at 34 degrees C with 85 +/- 5% relative humidity and then exposed to either 15 or 60 min of 8% O2. The rats were killed and brains perfusion fixed 5 days post-hypoxia for histological analysis. Rats exposed to 15 min of hypoxia-ischaemia suffered no mortality and 90% developed selective neuronal loss in the frontoparietal cortex and hippocampal CA1 region of the ipsilateral hemisphere. Rats exposed to 60 min of hypoxia consistently developed cortical pannecrosis and cellular loss in the striatum, thalamus, hippocampus and dentate gyrus of the ipsilateral hemisphere. The advantage of these models were ease of preparation, consistent neuronal loss scores and low mortality. The ability to induce either selective neuronal loss or pannecrosis provides an opportunity to investigate the mechanisms of damage or recovery associated with each pattern of cell loss.