Ernest Sirimanne

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.

Neuronal death and survival in two models of hypoxic-ischemic brain damage.

Two unilateral hypoxic-ischemia (HI) models (moderate and severe) in immature rat brain have been used to investigate the role of various transcription factors and related proteins in delayed neuronal death and survival. The moderate HI model results in an apoptotic-like neuronal death in selectively vulnerable regions of the brain while the more severe HI injury consistently produces widespread necrosis resulting in infarction, with some necrosis resistant cell populations showing evidence of an apoptotic type death. In susceptible regions undergoing an apoptotic-like death there was not only a prolonged induction of the immediate early genes, c-jun, c-fos and nur77, but also of possible target genes amyloid precursor protein (APP751) and CPP32. In contrast, increased levels of BDNF, phosphorylated CREB and PGHS-2 were found in cells resistant to the moderate HI insult suggesting that these proteins either alone or in combination may be of importance in the process of neuroprotection. An additional feature of both the moderate and severe brain insults was the rapid activation and/or proliferation of glial cells (microglia and astrocytes) in and around the site of damage. The glial response following HI was associated with an upregulation of both the CCAAT-enhancer binding protein alpha (microglia only) and NFkappaB transcription factors.

The role of the cyclic AMP-responsive element binding protein (CREB) in hypoxic-ischemic brain damage and repair.

The cyclic AMP-responsive element binding protein (CREB) is a basally expressed, post-translationally activated transcription factor that has been implicated in the trans-activation of a number of genes in response to cAMP and calcium signals. A unilateral hypoxic-ischemic (HI) injury in the 21 day old rat was used to examine a potential role for CREB (phosphorylated and unphosphorylated) in neuronal programmed cell death or cell survival. The selectively vulnerable CAI pyramidal cells, which undergo delayed neuronal death following mild HI, show a loss of CREB and phosphorylated CREB (pCREB) immunoreactivity on the injured side 48 and 72 h following HI. In contrast the resistant dentate granule cells and cortical cells produce a bimodal increase in pCREB immunoreactivity, peaking 6 and 48 h following HI. The fact that cells surviving the HI insult are showing increased activation of CREB suggests that this protein might be involved in the process of neuroprotection.

Loss of Ref-1 protein expression precedes DNA fragmentation in apoptotic neurons

Ref-1 is a bifunctional protein that has been implicated in the transcriptional regulation of AP-1 elements and in DNA repair. To investigate whether Ref-1 is involved in programmed cell death its expression was measured in the 21-day-old rat brain at various time-points following a moderate unilateral hypoxic-ischemic (HI) insult. The CA1 pyramidal cells, which are selectively vulnerable to HI injury, showed a significant decrease in Ref-1 immunoreactivity 48 h-7 days post-insult. This loss of Ref-1 immunoreactivity may contribute to a decrease in endogenous repair activity and the development of apoptosis in the CA1 pyramidal cells.

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.

ATF-2 phosphorylation in apoptotic neuronal death

Activating transcription factor (ATF-2) is a basic region-leucine zipper transcription factor that can mediate a diverse range of transcriptional responses including those generated by various forms of cellular stress. Activation of ATF-2 in response to these stimuli requires post-translational modification, in particular the phosphorylation of Thr69 and Thr71. To investigate whether ATF-2 activation also has a role in neuronal apoptosis, immunocytochemistry using a phospho-specific ATF-2 (Thr71) antibody was carried out in the 21 day old rat brain following a unilateral hypoxic-ischemic (HI) insult and PC12 cells cultured in the presence of okadaic acid. In both models a dramatic increase in phosphorylated ATF-2 was found within cells undergoing apoptosis.

Induction of clusterin in the immature brain following a hypoxic-ischemic injury.

A unilateral hypoxic-ischemic (HI) insult in the 21 day old rat has been used to assess the role of clusterin in nerve cell death. Both clusterin mRNA and protein levels were measured at various time points after moderate (15 min) and severe (60 min) HI insult using in situ hybridisation and immunocytochemistry respectively. The severe HI insult lead primarily to necrotic neuronal death and showed very little if any clusterin mRNA and protein induction on the ligated side of the brain. However, following the moderate HI insult there was a dramatic time-dependent accumulation of clusterin protein in neurons of the CA1-CA2 pyramidal cell layers in the hippocampus and cortical layers 3-5, regions undergoing delayed neuronal death. Clusterin mRNA expression, in contrast to neuronal protein accumulation, appeared to be glial in origin (probably astrocytes) with increases in mRNA in and around the hippocampal fissure and only a weak signal over the CA1-CA2 pyramidal cell layer. These results support the hypothesis that the clusterin protein is synthesised in the astrocytes, secreted and then taken up by dying neurons. Clusterin immunoreactivity and in situ DNA end-labelling performed on the same sections revealed that clusterin was accumulating in neurons destined to die by programmed cell death. However the relative time-courses of DNA fragmentation and clusterin immunoreactivity suggest that clusterin production was a result of the selective delayed neuronal death rather than being involved in the biochemical cascade of events that cause it.

Prostaglandin H synthase-2 and cytosolic phospholipase A2 in the hypoxic-ischemic brain: role in neuronal death or survival?

The breakdown of membrane phospholipids and subsequent arachidonic acid metabolism to prostanoids is a well-documented brain response to cerebral ischemia. To further elucidate the components of this signal transduction pathway, immunocytochemistry was used to determine the levels of two potentially important enzymes, cytosolic phospholipase A2 (cPLA2) and prostaglandin H synthase-2 (PGHS-2), in the immature rat brain following moderate unilateral hypoxic-ischemia (HI). The CA1 pyramidal cells of the hippocampus which undergo delayed neuronal death on the injured side following HI demonstrated a significant induction of PGHS-2 immunoreactivity 48 h post-insult. However, a consistent increase in PGHS-2 was also evident in the resistant dentate granule cells at an earlier time point. Although PGHS-2 is present in both susceptible and resistant cell populations following HI, the possibility remains that divergence further down-stream in the pathway is responsible for selective vulnerability. In contrast to the neuronal PGHS-2 expression, cPLA2 immunoreactivity appears to be of glial origin with increases in and around the CAI-2 pyramidal cell layer at the 72-168-h time points. These results suggest that prostanoids are likely to serve important roles in HI brain damage and repair in infant brain.

Do c-Jun, c-Fos, and amyloid precursor protein play a role in neuronal death or survival?

A unilateral hypoxic‐ischemic (HI) episode in immature rat brain was used to investigate the role of the immediate early genes c‐fos and c‐jun in delayed neuronal death and survival. This HI paradigm results in an apoptotic cell death in selectively vulnerable areas, in particular the hippocampal CA1 pyramidal cell layer. In susceptible regions undergoing delayed neuronal death there was a prolonged induction of both c‐Jun and c‐Fos (mRNA and protein). This expression occurred in parallel with a pronounced increase in AP‐1 DNA binding activity but was not associated with either increased levels of Jun NH2‐terminal kinase or phosphorylation of c‐Jun (ser‐63). In addition to changes in immediate early gene expression, the CA1 neurons showed a delayed increase in the expression of amyloid precursor protein (APP751) mRNA, suggesting that APP, which contains an AP‐1 site, might be a down‐stream gene regulated by the Jun transcription factor in neurons dying by apoptosis. The surviving dentate granule cells also showed an increase in Fos, Jun, and APP751 although this expression occurred earlier than in the CA1 neurons and declined rapidly. These results are discussed with respect to the role of these proteins in neuronal death and survival. J. Neurosci. Res. 53:330–342, 1998.

CCAAT-enhancer binding proteinα is expressed in activated microglial cells after brain injury

Abstract Microglial cells play important roles in brain injury and repair and are implicated in diseases such as Alzheimers disease, Creutzfeldt–Jacob disease, multiple sclerosis, the Aids Dementia Complex and stroke. Despite their importance in neuropathology, the underlying molecular basis for the activation of microglia after brain injury is not understood. We show, using RT-PCR, in situ hybridisation, immunocytochemistry, and electrophoretic mobility shift assay, that the CCAAT-enhancer binding proteinα (C/EBPα), a sequence specific DNA-binding protein, is induced in microglial cells, but not astrocytes or neurons, after hypoxic–ischemic brain injury. These results suggest that C/EBPα might regulate gene expression and consequentially have a role in the activation and/or proliferation of microglia following brain injury.