Brain vulnerability and viability after ischaemia.

The susceptibility of the brain to ischaemic injury dramatically limits its viability following interruptions in blood flow. However, data from studies of dissociated cells, tissue specimens, isolated organs and whole bodies have brought into question the temporal limits within which the brain is capable of tolerating prolonged circulatory arrest. This Review assesses cell type-specific mechanisms of global cerebral ischaemia, and examines the circumstances in which the brain exhibits heightened resilience to injury. We suggest strategies for expanding such discoveries to fuel translational research into novel cytoprotective therapies, and describe emerging technologies and experimental concepts. By doing so, we propose a new multimodal framework to investigate brain resuscitation following extended periods of circulatory arrest.

Neuron-to-astrocyte signaling is central to the dynamic control of brain microcirculation.

The cellular mechanisms underlying functional hyperemia--the coupling of neuronal activation to cerebral blood vessel responses--are not yet known. Here we show in rat cortical slices that the dilation of arterioles triggered by neuronal activity is dependent on glutamate-mediated [Ca(2+)](i) oscillations in astrocytes. Inhibition of these Ca(2+) responses resulted in the impairment of activity-dependent vasodilation, whereas selective activation--by patch pipette--of single astrocytes that were in contact with arterioles triggered vessel relaxation. We also found that a cyclooxygenase product is centrally involved in this astrocyte-mediated control of arterioles. In vivo blockade of glutamate-mediated [Ca(2+)](i) elevations in astrocytes reduced the blood flow increase in the somatosensory cortex during contralateral forepaw stimulation. Taken together, our findings show that neuron-to-astrocyte signaling is a key mechanism in functional hyperemia.

Differences in clot preparation determine outcome of recombinant tissue plasminogen activator treatment in experimental thromboembolic stroke.

BACKGROUND AND PURPOSE: Thrombin-induced clots used in experimental thromboembolic stroke differ from clots forming spontaneously under clinical conditions. We investigated whether this difference influences the efficacy and outcome of thrombolytic treatment. METHODS: In rats, the middle cerebral artery was occluded by intracarotid injection of fibrin-rich clots, prepared either according to established methods by adding thrombin to freshly drawn arterial blood or by spontaneous coagulation. The mechanical properties of clots were determined in vitro by elasticity and plasticity tests. One hour after embolism, thrombolysis was started by intra-arterial application of recombinant tissue plasminogen activator (rtPA) (10 mg/kg). Treatment efficacy was monitored by MR measurements of blood perfusion, apparent diffusion coefficient (ADC), T2 relaxation time and blood-brain barrier permeability, and by pictorial measurements of ATP and pH. RESULTS: Thrombin-induced clots were classified as elastic, and spontaneously forming clots were classified as plastic. Middle cerebral artery embolism with thrombin-induced or spontaneously forming clots led to similar reduction of perfusion and ADC, but rtPA treatment efficacy differed greatly. In the spontaneously forming clot group, blood perfusion returned to or above control within 2 hours, ADC and ATP normalized, tissue pH exhibited alkalosis, and T2 and blood-brain barrier permeability did not change. In the thrombin-induced clot group, in contrast, blood reperfusion was delayed, ADC and ATP remained reduced, tissue pH was acidic, and edema developed, as reflected by increased T2 and blood-brain barrier permeability. CONCLUSIONS: rtPA-induced thrombolysis promotes rapid reperfusion and tissue recovery in animals embolized with spontaneously forming clots but not in those embolized with thrombin-induced clots. This difference is explained by the different mechanical and possibly molecular consequences of clot preparation and must be considered for the interpretation of thrombolysis experiments.

Thrombolytic treatment of clot embolism in rat: comparison of intra-arterial and intravenous application of recombinant tissue plasminogen activator.

BACKGROUND AND PURPOSE: We sought to test the hypothesis that intra-arterial recombinant tissue plasminogen activator (rtPA) treatment of thromboembolic stroke is more efficient than intravenous application. METHODS: Rats were embolized by intracarotid injection of autologous fibrin-rich blood clots. One hour later rtPA (10 mg/kg) was infused either intravenously (n=8) or intra-arterially (n=8). Control rats (n=8) received intra-arterial infusion of saline. Treatment was monitored by MR perfusion-weighted imaging and apparent diffusion coefficient (ADC) imaging, and outcome was evaluated by comparing incidence of hemorrhages and lesion volumes of ATP and pH. RESULTS: Clot embolism led to a decline of perfusion-weighted imaging signal intensity in the middle cerebral artery territory to <40% of control. Both intra-arterial and intravenous treatment significantly improved blood flow in cerebral cortex but not in caudate putamen. In untreated animals, ATP and pH lesion volumes were 510.3+/-94.5 and 438.6+/-39.2 mm(3) at 7 hours after clot embolism, respectively. Both intravenous and intra-arterial rtPA treatment produced hemorrhagic complications but reduced ATP lesion size to 296.2+/-136.1 and 370.3+/-103.7 mm(3) and reduced pH lesion size to 263.3+/-114.6 and 303.3+/-103.0 mm(3), respectively (P<0.05 for untreated versus treated rats; no difference between intravenous and intra-arterial treatment). ADC imaging revealed that lesion reduction was due to inhibition of infarct growth but not to reversal of primary injury. CONCLUSIONS: This study documents reduction of injury by rtPA treatment but does not reveal a difference between intra-arterial and intravenous application. Our data do not support an advantage of intra-arterial thrombolysis.

Host-dependent tumorigenesis of embryonic stem cell transplantation in experimental stroke.

The therapeutical potential of transplantation of undifferentiated and predifferentiated murine embryonic stem cells for the regeneration of the injured brain was investigated in two rodent stroke models. Undifferentiated embryonic stem cells xenotransplanted into the rat brain at the hemisphere opposite to the ischemic injury migrated along the corpus callosum towards the damaged tissue and differentiated into neurons in the border zone of the lesion. In the homologous mouse brain, the same murine embryonic stem cells did not migrate, but produced highly malignant teratocarcinomas at the site of implantation, independent of whether they were predifferentiated in vitro to neural progenitor cells. The authors demonstrated a hitherto unrecognized inverse outcome after xenotransplantation and homologous transplantation of embryonic stem cells, which raises concerns about safety provisions when the therapeutical potential of human embryonic stem cells is tested in preclinical animal models.

[Embryonic stem cell therapy in experimental stroke: host-dependent malignant transformation]. / Embrionális ossejt terápia kísérletes stroke-ban: gazdafüggo malignus transzformáció.

INTRODUCTION: Therapeutic application of embryonic stem cells in neurodegenerative disorders like stroke is widely investigated in preclinical animal models. AIM: The authors studied the therapeutic potential of murine embryonic stem cells in two rodent models of stroke. METHODS: Undifferentiated and predifferentiated stem cells were implanted into the non-ischemic hemisphere of mice and rats following focal brain ischemia. The brains were analysed by immunohistochemistry and histology. The in vitro differentiation of the cells was checked by immunocytochemistry and Western-blot. RESULTS: After xenotransplantation in rats undifferentiated cells migrated along the corpus callosum towards the ischemic injury. Later stem cells differentiated into neurons in the border zone of the lesion. In the homologous mouse brain, the same murine embryonic stem cells did not migrate, but produced highly malignant teratocarcinomas at the site of implantation, independent of whether they were predifferentiated in vitro to neural progenitor cells. These experiments demonstrated a hitherto unrecognized adverse outcome after xenotransplantation and homologous transplantation of embryonic stem cells. CONCLUSION: This observation raises serious concerns about safety provisions when the therapeutic potential of human embryonic stem cells is tested in preclinical animal models. The clinical trials are based on the positive outcome of the xenologous experiments.

Heterozygosity for the mutated X-chromosome-linked L1 cell adhesion molecule gene leads to increased numbers of neurons and enhanced metabolism in the forebrain of female carrier mice.

Mutations in the X-chromosomal L1CAM gene lead to severe neurological deficits. In this study, we analyzed brains of female mice heterozygous for L1 (L1+/-) to gain insights into the brain structure of human females carrying one mutated L1 allele. From postnatal day 7 onward into adulthood, L1+/- female mice show an increased density of neurons in the neocortex and basal ganglia in comparison to wild-type (L1+/+) mice, correlating with enhanced metabolic parameters as measured in vivo. The densities of astrocytes and parvalbumin immunoreactive interneurons were not altered. No significant differences between L1+/- and L1+/+ mice were seen for cell proliferation in the cortex during embryonic days 11.5-15.5. Neuronal differentiation as estimated by analysis of doublecortin-immunoreactive cortical cells of embryonic brains was similar in L1+/- and L1+/+ mice. Interestingly, at postnatal days 3 and 5, apoptosis was reduced in L1+/- compared to L1+/+ mice. We suggest that reduced apoptosis leads to increased neuronal density in adult L1+/- mice. In conclusion, L1+/- mice display an unexpected phenotype that is not an intermediate between L1+/+ mice and mice deficient in L1 (L1-/y), but a novel phenotype which is challenging to understand regarding its underlying molecular and cellular mechanisms.

Immunohistochemical analysis of protein expression after middle cerebral artery occlusion in mice.

The effect of transient focal cerebral ischemia on protein regulation was studied in mice using multiparametric immunohistochemistry. Injury was characterized by measurements of blood flow, regional protein synthesis and terminal transferase biotinylated-dUTP nick end labeling (TUNEL). The proteins studied were selected from a previously established list of differentially regulated proteins and included the GTPases dynamin, RhoB, CAS and Ran BP-1, the transcription factors Nurr1 and p-Stat 6, the protein kinase MAPK p49, the splicing factors SRPK1 and hPrp16, the cell cycle control proteins cyclin B1 and Nek2, the inflammatory proteins FKBP12 and Rag2, the cell adhesion protein paxillin and the folding protein TCP-1. Regulation patterns were diverse and comprised ipsi- and/or contralateral up- and down-regulation with or without topical association to impeding cell death. Some proteins (SRPK1, TCP-1 and Nurr1) also exhibited post-ischemic translocation from the nucleus to the cytosol. Our observations stress the importance of regional analysis for the interpretation of proteomic data, and contribute to the identification of new pathways that may be involved in the evolution of post-ischemic brain injury.