Anders Lewén

Evidence of Phosphorylation of Akt and Neuronal Survival after Transient Focal Cerebral Ischemia in Mice

The serine-threonine kinase, Akt, prevents apoptosis by phosphorylation at serine-473 in several cell systems. After phosphorylation, activated Akt inactivates other apoptogenic factors, such as Bad or caspase-9, thereby inhibiting cell death. The present study examined phosphorylation of Akt at serine-473 and DNA fragmentation after transient focal cerebral ischemia in mice subjected to 60 minutes of focal cerebral ischemia by intraluminal blockade of the middle cerebral artery. Phospho-Akt was analyzed by immunohistochemistry and Western blot analysis. The DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end-labeling (TUNEL). Immunohistochemistry showed the expression of phospho-Akt was markedly increased in the middle cerebral artery territory cortex at 4 hours of reperfusion compared with the control, whereas it was decreased by 24 hours. Western blot analysis showed a significant increase of phospho-Akt 4 hours after focal cerebral ischemia in the cortex, whereas phospho-Akt was decreased in the ischemic core. Double staining with phospho-Akt and TUNEL showed different cellular distributions of phospho-Akt and TUNEL-positive staining. Phosphorylation of Akt was prevented after focal cerebral ischemia by LY294002, a phosphatidylinositol 3-kinase inhibitor, which facilitated subsequent DNA fragmentation. These results suggest that phosphorylation of Akt may be involved in determining cell survival or cell death after transient focal cerebral ischemia.

Effects of global ischemia duration on neuronal, astroglial, oligodendroglial, and microglial reactions in the vulnerable hippocampal CA1 subregion in rats

The hippocampal CA1 neurons are selectively vulnerable to global ischemia, and neuronal death occurs in a delayed manner. The threshold of global ischemia duration that induces neuronal death has been studied, but the relationship between ischemia duration and glial death in the hippocampal CA1 area has not been fully studied. We examined neuronal/glial viability and morphological changes in the CA1 subregion after different durations of global ischemia. Global ischemia was induced in Sprague-Dawley rats by 10, 5, and 3 min of bilateral common carotid artery occlusion and hypotension. At 1-56 days after ischemia, the morphological reactions of neurons, astrocytes, oligodendrocytes, and microglia were immunohistochemically evaluated. Most of the hippocampal CA1 pyramidal neurons underwent delayed death at 3 days after 10/5 min of ischemia, but not after 3 min of ischemia. The number of astrocytes gradually declined after 10/5 min of ischemia, and viable astrocytes showed characteristic staged morphological reactions. Oligodendrocytes also showed morphological changes in their processes after 10/5 min of ischemia. Microglia transformed into a reactive form at 5 days only after 10/5 min of ischemia. These data suggest that some morphological changes in glial cells were not dependent on neuronal cell death, but their own reactions to the different severity of ischemia.

Distinct cellular patterns of upregulated chemokine expression supporting a prominent inflammatory role in traumatic brain injury

Cerebral gene expressions change in response to traumatic brain injury (TBI), and future trauma treatment may improve with increased knowledge about these regulations. We subjected C57BL/6J mice to injury by controlled cortical impact (CCI). At various time points post-injury, mRNA from neocortex and hippocampus was isolated, and transcriptional alterations studied using quantitative real-time polymerase chain reaction (PCR) and gene array analysis. Spatial distribution of enhanced expression was characterized by in situ hybridization. Products of the upregulated transcripts serve functions in a range of cellular mechanisms, including stress, inflammation and immune responses, and tissue remodeling. We also identified increased transcript levels characterizing reactive astrocytes, oligodendrocytes, and microglia, and furthermore, we demonstrated a novel pattern of scattered cell clusters expressing the chemokine Cxcl10. Notably, a sustained increase in integrin alpha X (Itgax), characterizing antigen-presenting dendritic cells, was found with the transcript located to similar cell clusters. In contrast, T-cell receptor alpha transcript showed only a modest increase. The induced P-selectin (Selp) expression level in endothelial cells, and chemokines from microglia, may guide perivascular accumulation of extravasating inflammatory monocytes differentiating into dendritic cells. In conclusion, our study shows that following TBI, secondary injury chiefly involves inflammatory processes and chemokine signaling, which comprise putative targets for pharmaceutical neuroprotection.

Glycerol as a marker for post-traumatic membrane phospholipid degradation in rat brain.

DEGRADATION of membrane phospholipids (PLs) is a well known phenomenon in acute brain injuries and is thought to underlie the disturbance of vital cellular membrane functions. In the present study glycerol, an end product of PL degradation, was examined in brain interstitial fluid as a marker of PL breakdown following experimental traumatic brain injury (TBI) using microdialysis. TBI was induced in artificially ventilated rats using the weight-drop technique. The trauma caused a significant, eight-fold increase of dialysate glycerol in the injured cortex, with the peak concentration in the second 10 min fraction after trauma. Glycerol then levelled off but remained significantly above sham-operated controls for the entire 4 h observation period in the perimeter of the injury region where scattered neuronal death is seen. The results support the concept that PL degradation occurs early after TBI and that interstitial glycerol, harvested by microdialysis, may be useful as a marker allowing in vivo monitoring of PL breakdown.

Changes in microtubule-associated protein 2 and amyloid precursor protein immunoreactivity following traumatic brain injury in rat: influence of MK-801 treatment

We investigated by immunohistochemistry dendritic and axonal changes occurring in the rat brain after mild focal cortical trauma produced by the weight drop technique. One and 3 days after injury, nerve cell bodies and dendrites in the perimeter of the impact site displayed decreased microtubule-associated protein 2 (MAP2) immunoreactivity. Some dendrites in the immediate adjacent region were more intensely stained and distorted. The dentate hilar region of the hippocampus showed a reduction of immunoreactive nerve cell bodies and dendrites. Twenty-one days after injury the strongly stained cortical dendrites and the reduction of immunoreactivity in the hippocampus remained, whereas the reduced staining in the perimeter of the lesion had normalised. These results indicate that there is a long-lasting disturbed dendritic organisation implicating impaired neurotransmission after this type of mild brain trauma. beta-Amyloid precursor protein (APP) immunohistochemistry revealed numerous stained axons in the ipsilateral subcortical white matter and thalamus indicating local and remote axonal injuries with disturbed axonal transport. Twenty-one days after injury, numerous small immunostained profiles appeared in the neuropil of the cortical impact site and in the ipsilateral thalamus. The axonal changes indicate disturbed connectivity between the site of the impact and other brain regions, chiefly the thalamus. The presence of beta-amyloid was investigated 21 days after trauma. There were no signs of beta-amyloid depositions in the brain after injury. Finally, we tested if the non-competitive NMDA receptor antagonist dizocilpine maleate (MK-801) could influence the observed MAP2 and APP changes. Pretreatment with this compound did not affect the early MAP2 and APP alterations. Instead, an increased expression of the APP antigen in the thalamus was observed 21 days after trauma in the MK-801-treated animals. The cause of this phenomenon is not known but may be related to a delayed neurotoxic action of MK-801 treatment.

Overexpression of SOD1 protects vulnerable motor neurons after spinal cord injury by attenuating mitochondrial cytochrome c release

Defective Cu, Zn‐superoxide dismutase (SOD1) is responsible for some types of amyotrophic lateral sclerosis, and ventral horn motor neurons (VMN) have been shown to die through a mitochondria‐dependent apoptotic pathway after chronic exposure to high levels of reactive oxygen species (ROS). VMN are also selectively vulnerable to mild spinal cord injury (SCI); however, the involvement of SOD1, ROS, and apoptosis in their death has not been clarified. Mild compression SCI was induced in SOD1‐overexpressing transgenic rats and wild‐type littermates. Superoxide production, mitochondrial release of cytochrome c, and activation of caspase‐9 were examined, and apoptotic DNA injury was also characterized. In the wild‐type animals, increased superoxide production, mitochondrial release of cytochrome c, and cleaved caspase‐9 were observed exclusively in VMN after SCI. Subsequently, a majority of VMN (75%) selectively underwent delayed apoptotic cell death. Transgenic animals showed less superoxide production, mitochondrial cytochrome c release, and caspase‐9 activation, resulting in death of only 45% of the VMN. These results suggest that the ROS‐initiated mitochondrial signaling pathway possibly plays a pivotal role in apoptotic VMN death after SCI and that increased levels of SOD1 in VMN reduce oxidative stress, thereby attenuating the activation of the pathway and delayed cell death.

Traumatic brain injury in rat produces changes of β-amyloid precursor protein immunoreactivity

beta-Amyloid precursor protein immunoreactivity (APP) was studied after a mild compression contusion trauma to rat parietal cortex. Neurones in the periphery of the cortical lesion, i.e. tissue subjected to shear stress, showed markedly reduced immunoreactivity 1 and 3 days after injury. Numerous axons in the ipsilateral subcortical white matter and thalamus became immunoreactive. At 21 days, small rounded profiles appeared in the neuropil of the damaged cortex and in the thalamus. Thus, traumatic brain injury appears to induce several types of APP changes. The accumulation in neuronal processes is probably caused by disturbed axonal transport induced by trauma. Since APP is assumed to be excitoprotective, modulating intracellular Ca2+ responses, the decreased immunoreactivity noticed in the periphery of the lesion may render the neurones in this region more vulnerable to secondary injury mechanisms.

The cytosolic antioxidant, copper/zinc superoxide dismutase, attenuates blood–brain barrier disruption and oxidative cellular injury after photothrombotic cortical ischemia in mice

Oxidative stress has been associated with the development of blood-brain barrier disruption and cellular injury after ischemia. The cytosolic antioxidant, copper/zinc superoxide dismutase, has been shown to protect against blood-brain barrier disruption and infarction after cerebral ischemia-reperfusion. However, it is not clear whether copper/zinc superoxide dismutase can protect against evolving ischemic lesions after thromboembolic cortical ischemia. In this study, the photothrombotic ischemia model, which is physiologically similar to thromboembolic stroke, was used to develop cortical ischemia. Blood-brain barrier disruption and oxidative cellular damage were investigated in transgenic mice that overexpress copper/zinc superoxide dismutase and in littermate wild-type mice after photothrombotic ischemia, which was induced by both injection of erythrosin B (30 mg/kg) and irradiation using a helium neon laser for 3 min. Free radical production, particularly superoxide, was increased in the lesioned cortex as early as 4 h after ischemia using hydroethidine in situ detection. The transgenic mice showed a prominent decrease in oxidative stress compared with the wild-type mice. Blood-brain barrier disruption, evidenced by quantitation of Evans Blue leakage, occurred 1 h after ischemia and gradually increased up to 24 h. Compared with the wild-type mice, the transgenic mice showed less blood-brain barrier disruption, a decrease in oxidative DNA damage using 8-hydroxyguanosine immunohistochemistry, a subsequent decrease in DNA fragmentation using the in situ nick-end labeling technique, and decreased infarct volume after ischemia. From these results we suggest that superoxide anion radical is an important factor in blood-brain barrier disruption and oxidative cellular injury, and that copper/zinc superoxide dismutase could protect against the evolving infarction after thromboembolic cortical ischemia.

Regulation of pituitary adenylate cyclase activating polypeptide and its receptor type 1 after traumatic brain injury : Comparison with brain-derived neurotrophic factor and the induction of neuronal cell death

Neurotrophic factors are known to promote neuronal survival during development and after acute brain injury. Recent data suggest that some neuropeptides also exhibit neurotrophic activities, as shown for the pituitary adenylate cyclase activating polypeptide, which increases the survival of various neuronal populations in culture. Employing in situ hybridization techniques, we have studied the regulation of messenger RNA for pituitary adenylate cyclase activating polypeptide and its receptor type 1 after a moderate traumatic brain injury to rat brain cortex. We have further compared their messenger RNA expression to that of brain-derived neurotrophic factor and to the amount of cell death occurring in the brain at various times after the brain injury. Levels of brain-derived neurotrophic factor messenger RNA increased rapidly within 2 h after trauma in cortex and hippocampus, and returned to control levels thereafter. The levels of messenger RNA for pituitary adenylate cyclase activating polypeptide also increased with time in the injured brains and reached maximal expression at 72 h, i.e. the end of the observation period. The alterations in pituitary adenylate cyclase activating polypeptide messenger RNA levels were particularly pronounced in the perifocal region and in the ipsilateral dentate gyrus of the brain injury. In contrast, the messenger RNA levels encoding pituitary adenylate cyclase activating polypeptide receptor type 1 first decreased after trauma and were then normalized in the dentate gyrus. There was a large increase in the number of cells labelled for DNA breaks at 12 h post-trauma, indicative of enhanced cell death. The number of labelled cells, however, decreased at later stages concomitant with an increase in the expression of pituitary adenylate cyclase activating polypeptide messenger RNA. Pituitary adenylate cyclase activating polypeptide rescued cortical neurons in cultures against ionomycin-induced cell death, supporting the concept of a neuroprotective effect for the peptide. These results demonstrate a differential regulation of messenger RNA for brain-derived neurotrophic factor and the pituitary adenylate cyclase activating polypeptide and its receptor after brain trauma. The data also suggest that pituitary adenylate cyclase activating polypeptide might have a beneficial effect in brain injury by counteracting neuronal cell death.

Oxidative stress-dependent release of Mitochondrial cytochrome c after traumatic brain injury

Mitochondrial cytochrome c translocation to the cytosol initiates the mitochondrial-dependent apoptotic pathway. This event has not been previously reported in traumatic brain injury (TBI). The authors determined the expression of cytochrome c in cytosolic and mitochondrial fractions after severe TBI produced by the controlled cortical impact model in the mouse. One hour after trauma there was an increase in cytosolic cytochrome c immunoreactivity. The increases in cytosolic cytochrome c preceded DNA fragmentation, which started at 4 hours. Western blots of mitochondrial and cytosolic fractions confirmed that there was a translocation of cytochrome c from the mitochondria after TBI. Mice deficient in manganese superoxide dismutase (MnSOD) showed an increased loss of mitochondrial cytochrome c after trauma, but less apoptotic cell death 4 and 24 hours after injury compared with wild-type control mice. However, the overall cell death was increased in MnSOD mice, as illustrated by a larger cortical lesion in these animals. The results show that cytochrome c is released from the mitochondria after severe TBI partly by a free radical–dependent mechanism, and that massive mitochondrial cytochrome c release is a predictor of necrotic cell death rather than apoptosis.