MEK-inhibitor U0126 in hyperglycaemic focal ischaemic brain injury in the rat.

BACKGROUND: Hyperglycaemia aggravates ischaemic brain injury, possibly due to activation of signalling pathways involving mitogen-activated protein kinases (MAPK). In this study, the activation of MAPK/ERK was inhibited using the upstream inhibitor of MAPK-ERK-kinase (MEK) U0126, and the effects on focal brain ischaemia were evaluated during normo- and hyperglycaemia. MATERIALS AND METHODS: Temporary (90 min) middle cerebral artery occlusion (MCAO) was induced in five groups of rats. U0126 (400 microg kg(-1)) or vehicle was given as 60-min intravenous infusions starting either 30 min prior to MCAO or 30 min prior to reperfusion. The infarct size was determined by perfusion with tetrazolium red after 24 h of survival, and the neurology was tested with the 4-level scale of Bederson and performance on an inclined plane. The inhibitory effect on the targeted MEK enzyme was investigated by analysing the phosphorylation of the downstream target ERK with western immunoblotting. Two subgroups were investigated with magnetic resonance imaging (MRI), including diffusion-weighted (DWI) and perfusion-weighted imaging (PWI). RESULTS: U0126 effectively reduced the infarct size and improved neurology in hyperglycaemic rats both when given before and after ischemic onset. This effect was not accompanied by any detectable changes in cerebral blood flow on MRI. Normoglycaemic rats had generally milder injuries compared with the hyperglycaemic and there was a nonsignificant trend for U0126 to reduce damage also in the nonhyperglycaemic groups. CONCLUSIONS: In conclusion, U0126 appears to be neuroprotective in this model of hyperglycaemic ischaemic brain injury. The findings support the pathogenic importance of the MEK-ERK pathway in hyperglycaemic-ischaemic brain injury.

Blood glucose in acute stroke, different therapeutic targets for diabetic and non-diabetic patients?

OBJECTIVES: To study the impact of blood glucose concentrations on early stroke mortality in diabetic and non-diabetic stroke patients, and to identify the optimal blood glucose concentration for each patient category. MATERIAL AND METHODS: A representative sample of 81 diabetic and 366 non-diabetic stroke patients was studied. Logistic regression analyses were performed in order to estimate the impact of blood glucose concentrations on admission and during hospital stay and other clinical parameters on 30-day case-fatality. Receiver operating characteristic curves were used to predict case-fatality by blood glucose. RESULTS: Blood glucose, body temperature and level of consciousness were independently related to early stroke mortality in diabetic and non-diabetic patients. The mean blood glucose concentration had a greater impact on 30-day case-fatality than the admission blood glucose, particularly in diabetic patients. A mean blood glucose concentration above 10.3 mmol/l predicted 30-day case-fatality in diabetic patients. The corresponding value was 6.3 mmol/l in non-diabetic patients. CONCLUSION: Improved blood glucose control has a potential to reduce early stroke mortality. The optimal glucose concentration seems to be higher in diabetic than in non-diabetic patients.

Differential early mitogen-activated protein kinase activation in hyperglycemic ischemic brain injury in the rat.

BACKGROUND: Hyperglycemia aggravates brain injury induced by focal ischemia-reperfusion. The mitogen-activated protein kinase (MAPK) members extracellular-signal regulated kinase (Erk) and c-Jun N-terminal kinase (JNK) have been proposed as mediators of ischemic brain injury, and Erk is strongly activated by combined hyperglycemia and transient global ischemia. It is unclear whether similar MAPK activation appears in focal brain ischemia with concomitant hyperglycemia. DESIGN: Hyperglycemia was induced in rats by an intraperitoneal bolus of glucose (2 g kg(-1)). The rats were then subjected to 90 min of transient middle cerebral artery occlusion (MCAO). Erk and JNK activation were investigated with immunofluorescence and Western blot along with infarct size measurement based on tetrazolium staining and neurological score. RESULTS: The hyperglycemic rats showed increased tissue damage and impaired neurological performance after 1 day compared with controls. The hyperglycemia was generally moderate (< 15 mM). Erk activation was increased after 30 min of reperfusion in the ischemic cortex of the hyperglycemic rats, while JNK activation was present on the contralateral side. Phospho-Erk immunofluorescence revealed marked neuronal activation of Erk in the ischemic cortex of hyperglycemic rats compared with controls. CONCLUSION: Besides confirming the detrimental effects of hyperglycemia on focal ischemia-reperfusion, this study shows that hyperglycemia strongly activates the pathogenic mediator Erk in the ischemic brain in the early phase of reperfusion. JNK activation at this stage is present in the nonischemic hemisphere. The functional relevance of these findings needs further investigation.

Recruitment of type I collagen producing cells from the microvasculature in vitro.

We have previously suggested that microvascular pericytes can differentiate into fibroblast-like, type I collagen-producing cells during excessive dermal scarring in vivo (Sundberg, C., Ivarsson, M., Gerdin, B., and Rubin, K., Lab. Invest. 74, 454-468, 1996). Here we have investigated to what extent pericytes derived from microvessels of full-term human placenta exhibited this capacity in vitro. Vascular fragments of human term placenta were isolated by enzymatic digestion and separation in Percoll. Their microvascular origin was ascertained by confocal microscopy using antibodies specific for endothelial cells (PAL-E) and pericytes (high-molecular-weight-melanoma-associated antigen). When vascular fragments were cultured in vitro, large cells with irregular edges migrated out from the fragments. After 4-6 days in culture, these cells started to proliferate and reached near confluence after approximately 8 days. The cultures were not overgrown by clones of cells with a high proliferative capacity, as demonstrated by cell membrane fluorescence staining and Ki67 expression. Expression of PAL-E, high-molecular-weight-melanoma-associated antigen, smooth muscle alpha-actin, desmin, and collagen synthesis (prolyl-4-hydroxylase and type I procollagen, as well as collagen pro-alpha1(I) mRNA) were followed during a culture period of 8 days. The cells were PAL-E negative but expressed high-molecular-weight-melanoma-associated antigen, smooth muscle alpha-actin, and desmin. Based on morphology and expression of the various markers, the outgrowing cells were identified as pericytes. With time in culture the cells decreased their expression of all these markers and increased their expression of prolyl-4-hydroxylase, type I procollagen, and collagen pro-alpha1(I) mRNA. Metabolic labeling and SDS-PAGE analysis of labeled proteins revealed that type I collagen was the major collagen species synthesized in the cultures. Our results support the hypotheses that pericytes can leave the vasculature and differentiate into collagen-producing cells and that cultured "fibroblasts" are derived from pericytes.