Juha Yrjänheikki

Pyrrolidine dithiocarbamate inhibits translocation of nuclear factor kappa-B in neurons and protects against brain ischaemia with a wide therapeutic time window

Pyrrolidine dithiocarbamate (PDTC) is an antioxidant and inhibitor of transcription factor nuclear factor kappa‐B (NF‐κB). Because the role of NF‐κB in brain injury is controversial and another NF‐κB inhibiting thiocarbamate, DDTC, was recently shown to increase ischaemic brain damage, we investigated the effect of PDTC on transient brain ischaemia. Ischaemia was induced by occlusion of the middle cerebral artery (MCAO). In Wistar rats, the PDTC treatment started even 6 h after MCAO reduced the infarction volume by 48%. PDTC protected against MCAO also in spontaneously hypertensive rats and against forebrain ischaemia in Mongolian gerbils. PDTC prevented NF‐κB activation in the ischaemic brain as determined by reduced DNA binding and nuclear translocation of NF‐κB in neurons. PDTC had anti‐inflammatory effect by preventing induction of NF‐κB‐regulated pro‐inflammatory genes. In ischaemic rats, NF‐κB was localized in cyclo‐oxygenase‐2‐immunoreactive neurons. Blood cytokine levels were not altered by ischaemia or PDTC. When cultured neurons were exposed to an excitotoxin, no production of reactive oxygen species was detected, but PDTC provided protection and prevented nuclear translocation of NF‐κB. The clinically approved PDTC and its analogues may act as anti‐inflammatories and may be safe therapies in stroke with a wide time window.

Spreading Depression–Induced Gene Expression Is Regulated by Plasma Glucose

BACKGROUND AND PURPOSE Plasma glucose and spreading depression (SD) are both determinants of brain ischemia. The purpose of this study was to examine whether plasma glucose affects SD-induced gene expression in the cortex. METHODS SD was induced by topical application of KCl. Hyperglycemia and hypoglycemia were induced by intraperitoneal injection of glucose and insulin, respectively. The expression of c-fos, cyclooxygenase-2 (COX-2), protein kinase C-delta (PKCdelta), and heme oxygenase-1 (HO-1) was determined by in situ hybridization. RESULTS SD alone induced expression of c-fos (by 340%), COX-2 (210%), HO-1 (470%), and PKCdelta (410%). Hypoglycemia (2.4+/-0.9 mmol/L) alone did not induce gene expression, and hyperglycemia (22.1+/-3.7 mmol/L) alone induced only c-fos by 42%. When hypoglycemia was induced 30 minutes before SD, c-fos induction was enhanced by 145%, but the induction of HO-1 and PKCdelta was reduced to 43% and 64%, respectively. When hyperglycemia was induced 30 minutes before SD, c-fos induction was enhanced by 388% and COX-2 expression by 53%, whereas the induction of PKCdelta and HO-1 was reduced to 54% and 51%, respectively. The frequency, amplitude, and duration of direct current potentials were unaltered in hyperglycemic SD animals, whereas in hypoglycemic animals the duration was increased by 47%. CONCLUSIONS While SD induces expression of several genes, the availability of glucose regulates the extent of the gene induction. The effect of glucose is different on early-response genes (c-fos and COX-2) compared with late-response genes. Plasma glucose may contribute to neuronal damage partially by regulating gene expression.

Long-term protective effect of atorvastatin in permanent focal cerebral ischemia.

Statins exert beneficial effects in brain diseases including stroke. Here, we investigated whether oral prophylactic atorvastatin provides long-term neuroprotection and functional recovery in permanent middle cerebral artery occlusion (pMCAO), and whether cerebral hemodynamics are affected. Male Long-Evans rats were treated with 10 mg/kg oral atorvastatin for 14 days and subjected to pMCAO. Cerebral hemodynamics were measured by bolus tracking MRI and laser Doppler flowmetry (LDF). Infarct volume was quantified at 1 week by T2-MRI and at 3 weeks by histology. Rats were also subjected to neuroscoring and cylinder test. The number of animals per group was 10. The infarct volumes were 100.8 +/- 8.2 and 47.3 +/- 5.5 mm(3) in vehicle, and 68.7 +/- 11.0 and 28.6 +/- 3.82 mm(3) in atorvastatin group at 7 and 21 days post-ischemia, respectively (mean +/- SEM). Atorvastatin significantly reduced infarct volume both at 7 and 21 days (P = 0.04 and 0.03, respectively, 1-way ANOVA). Interestingly, no improvement in cerebral hemodynamic parameters was observed in atorvastatin treated animals. The vehicle group recovered normal neuroscore at day 13, whereas atorvastatin group recovered already at day 10 after pMCAO. All treatment groups preferred to use the unaffected forelimb for rearing in Cylinder test, whereas the defected forelimb use was minimal in all groups. These results suggest that oral atorvastatin protects cerebral tissue against the subsequent pMCAO without influencing cerebral hemodynamic parameters, and it may well be that persons with ongoing atorvastatin treatment benefit in the incidence of stroke.

Interleukin‐1 beta‐induced expression of the prostaglandin E2‐receptor subtype EP3 in U373 astrocytoma cells depends on protein kinase C and nuclear factor‐kappaB

Both interleukin‐1β (IL‐1β) and prostaglandins (PGs) are important mediators of physiological and pathophysiological processes in the brain. PGE2 exerts its effects by binding to four different types of PGE2 receptors named EP1–EP4. EP3 has found to be expressed in neurons, whereas expression of EP3 in glial cells has not been reported in the brain yet. Here we describe IL‐1β‐induced EP3 receptor expression in human astrocytoma cells, primary astrocytes of rat and human origin and in rat brain. Using western blot, we found a marked up‐regulation of EP3 receptor synthesis in human and rat primary glial cells. Intracerebroventricular administration of IL‐1β stimulated EP3 receptor synthesis in rat hippocampus. The analysis of involved signal transduction pathways by pathway‐specific inhibitors revealed an essential role of protein kinase C and nuclear factor‐κB in astrocytic IL‐1β‐induced EP3 synthesis. Our data suggest that PGE2 signaling in the brain may be altered after IL‐1β release due to up‐regulation of EP3 receptors. This might play an important role in acute and chronic conditions such as cerebral ischemia, traumatic brain injury, HIV‐encephalitis, Alzheimers disease and prion diseases in which a marked up‐regulation of IL‐1β is followed by a prolonged increase of PGE2 levels in the brain.

Cerebral Blood Volume Alterations in the Perilesional Areas in the Rat Brain after Traumatic Brain Injury—Comparison with Behavioral Outcome

In the traumatic brain injury (TBI) the initial impact causes both primary injury, and launches secondary injury cascades. One consequence, and a factor that may contribute to these secondary changes and functional outcome, is altered hemodynamics. The relative cerebral blood volume (CBV) changes in rat brain after severe controlled cortical impact injury were characterized to assess their interrelations with motor function impairment. Magnetic resonance imaging (MRI) was performed 1, 2, 4 h, and 1, 2, 3, 4, 7, and 14 days after TBI to quantify CBV and water diffusion. Neuroscore test was conducted before, and 2, 7, and 14 days after the TBI. We found distinct temporal profile of CBV in the perilesional area, hippocampus, and in the primary lesion. In all regions, the first response was drop of CBV. Perifocal CBV was reduced for over 4 days thereafter gradually recovering. After the initial drop, the hippocampal CBV was increased for 2 weeks. Neuroscore demonstrated severely impaired motor functions 2 days after injury (33% decrease), which then slowly recovered in 2 weeks. This recovery parallelled the recovery of perifocal CBV. CBV MRI can detect cerebrovascular pathophysiology after TBI in the vulnerable perilesional area, which seems to potentially associate with time course of sensory-motor deficit.

Induction of ornithine decarboxylase mRNA in transient focal cerebral ischemia in the rat

We used in situ hybridization to localize the long-term changes in ornithine decarboxylase (ODC) expression after a 90 min occlusion of the middle cerebral artery (MCAO) in the rat. The ODC mRNA was induced in the ipsilateral dentate gyrus (DG) and throughout the ischemic cortex at 12 h and still at 3 days after reperfusion. The induction was blocked by an N-methyl-D-aspartate (NMDA) receptor antagonist suggesting that ODC induction is NMDA receptor-mediated. The long-lasting up-regulation detected in regions where no cellular damage usually occurs, favors the hypothesis that ODC expression does not contribute to neuronal death after stroke.

Spreading depression-induced expression of c-fos and cyclooxygenase-2 in transgenic mice that overexpress human copper/zinc-superoxide dismutase

Spreading depression (SD) is a wave of sustained depolarization challenging the energy metabolism of cells without causing irreversible damage. SD is a major mechanism of gene induction that takes place in cortical injury, including ischemia. We studied the role of oxygen radicals in SD-induced c-fos and cyclooxygenase-2 (COX-2) induction using transgenic (Tg) mice that overexpress copper/zinc-superoxide dismutase (SOD1). The frequency, amplitude and duration of SD waves were similar in the Tg mice and wild-type littermates. c-fos and COX-2 mRNAs were strongly induced 1 and 4 h after SD. The induction of both genes was slightly but significantly less at 4 h in the Tg mice. The results indicate that even a mild, noninjurious metabolic stimulation increases the concentration of oxygen radicals to the level that contributes to gene expression.

Inflammation and Potential Anti-Inflammatory Approaches in Stroke

Ischemic stroke develops when a major brain artery, most commonly the middle cerebral artery, is obstructed by an embolus or thrombosis and, consequently, blood flow in a restricted area of the brain is severely reduced. Even though animal research conducted on acute ischemic injury has revealed several pathophysiological cascades, which contribute to the enlargement of brain infarction, no major breakthroughs in developing clinically relevant stroke therapy have been achieved. There are several reasons for failed translation of successful basic research approaches into effective treatment modalities for stroke in humans (1). One important factor is that the animal models do not reflect well enough the ischemic damage in both permanent (no reperfusion) and transient (with reperfusion) ischemia in aged female and male victims, which may suffer from other age-related diseases, such as hypertension, diabetes, and Alzheimer’s disease (2). Another, even a more valid, fact is that the therapeutic time window for treating human stroke is delayed beyond the hours after the insult and, therefore, the pathophysiological events targeted pharmacologically need to occur several hours or days later (2). For these reasons, targeting mechanisms such as various glutamate receptors and calcium channels may not be considered for treatment of most of the stroke patients.