Direct myosin-2 inhibition enhances cerebral perfusion resulting in functional improvement after ischemic stroke.

Acute ischemic stroke treatment faces an unresolved obstacle as capillary reperfusion remains insufficient after thrombolysis and thrombectomy causing neuronal damage and poor prognosis. Hypoxia-induced capillary constriction is mediated by actomyosin contraction in precapillary smooth muscle cells (SMCs) therefore smooth muscle myosin-2 could be an ideal target with potentially high impact on reperfusion of capillaries. Methods: The myosin-2 inhibitor para-aminoblebbistatin (AmBleb) was tested on isolated human and rat arterioles to assess the effect of AmBleb on vasodilatation. Transient middle cerebral artery occlusion (MCAO) was performed on 38 male Wistar rats followed by local administration of AmBleb into the ischemic brain area. Development of brain edema and changes in cerebrovascular blood flow were assessed using MRI and SPECT. We also tested the neurological deficit scores and locomotor asymmetry of the animals for 3 weeks after the MCAO operation. Results: Our results demonstrate that AmBleb could achieve full relaxation of isolated cerebral arterioles. In living animals AmBleb recovered cerebral blood flow in 32 out of the 65 affected functional brain areas in MCAO operated rats, whereas only 8 out of the 67 affected areas were recovered in the control animals. Animals treated with AmBleb also showed significantly improved general and focal deficit scores in neurological functional tests and showed significantly ameliorated locomotor asymmetry. Conclusion: Direct inhibition of smooth muscle myosin by AmBleb in pre-capillary SMCs significantly contribute to the improvement of cerebral blood reperfusion and brain functions suggesting that smooth muscle myosin inhibition may have promising potential in stroke therapies as a follow-up treatment of physical or chemical removal of the occluding thrombus.

Complement component 1q subcomponent binding protein in the brain of the rat.

Complement component 1q subcomponent binding protein (C1qbp) is a multifunctional protein involved in immune response, energy homeostasis of cells as a plasma membrane receptor, and a nuclear, cytoplasmic or mitochondrial protein. Recent reports suggested its neuronal function, too, possibly in axon maintenance, synaptic function, and neuroplasticity. Therefore, we addressed to identify C1qbp in the rat brain using in situ hybridization histochemistry and immunolabelling at light and electron microscopic level. C1qbp has a topographical distribution in the brain established by the same pattern of C1qbp mRNA-expressing and protein-containing neurons with the highest abundance in the cerebral cortex, anterodorsal thalamic nucleus, hypothalamic paraventricular (PVN) and arcuate nuclei, spinal trigeminal nucleus. Double labelling of C1qbp with the neuronal marker NeuN, with the astrocyte marker S100, and the microglia marker Iba1 demonstrated the presence of C1qbp in neurons but not in glial cells in the normal brain, while C1qbp appeared in microglia following their activation induced by focal ischemic lesion. Only restricted neurons expressed C1qbp, for example, in the PVN, magnocellular neurons selectively contained C1qbp. Further double labelling by using the mitochondria marker Idh3a antibody suggested the mitochondrial localization of C1qbp in the brain, confirmed by correlated light and electron microscopy at 3 different brain regions. Post-embedding immunoelectron microscopy also suggested uneven C1qbp content of mitochondria in different brain areas but also heterogeneity within single neurons. These data suggest a specific function of C1qbp in the brain related to mitochondria, such as the regulation of local energy supply in neuronal cells.

Selegiline promotes NOTCH-JAGGED signaling in astrocytes of the peri-infarct region and improves the functional integrity of the neurovascular unit in a rat model of focal ischemia.

PURPOSE: Our experiments aimed at exploring potential neurorestorative mechanisms of selegiline, a compound routinely used in the treatment of Parkinson's disease and previously shown to improve the functional recovery of stroke patients. METHODS: Selegiline was administered continuously via osmotic mini-pumps between 48 and 216 hours following middle cerebral artery occlusion (MCAO) in rats. Twenty-four hours before sacrifice, the animals underwent magnetic resonance imaging (MRI). After decapitation, the peri-infarct region was dissected to perform a TAQMAN array gene expression study, and brains were fixed for immunolabeling. RESULTS: In addition to the previously known induction of anti-apoptosis genes, selegiline significantly increased the mRNA level of Notch 1 receptor and its ligand Jagged 1. Immunohistochemistry demonstrated elevated Notch 1 and Jagged 1 immunoreactivities in the peri-infarct region. Double labeling with glial markers revealed that both Notch 1 and Jagged 1 were expressed in astrocytes but not in microglia. MRI examination indicated significantly reduced edema in selegiline-treated rats compared to control MCAO rats, and increased capillary network density was found in the peri-infarct region of the selegiline-treated animals. CONCLUSION: Our results suggest that selegiline treatment enhances Notch-Jagged signaling in astrocytes, reduces peri-lesional edema and potentially helps preserve the capillary network following focal ischemia.

Induction of transforming growth factor beta receptors following focal ischemia in the rat brain.

Transforming growth factor-ßs (TGF-ßs) regulate cellular proliferation, differentiation, and survival. TGF-ßs bind to type I (TGF-ßRI) and II receptors (TGF-ßRII), which are transmembrane kinase receptors, and an accessory type III receptor (TGF-ßRIII). TGF-ß may utilize another type I receptor, activin-like kinase receptor (Alk1). TGF-ß is neuroprotective in the middle cerebral artery occlusion (MCAO) model of stroke. Recently, we reported the expression pattern of TGF-ß1-3 after MCAO. To establish how TGF-ßs exert their actions following MCAO, the present study describes the induction of TGF-ßRI, RII, RIII and Alk1 at 24 h, 72 h and 1 mo after transient 1 h MCAO as well as following 24 h permanent MCAO using in situ hybridization histochemistry. In intact brain, only TGF-ßRI had significant expression: neurons in cortical layer IV contained TGF-ßRI. At 24 h after the occlusion, no TGF-ß receptors showed induction. At 72 h following MCAO, all four types of TGF-ß receptors were induced in the infarct area, while TGF-ßRI and RII also appeared in the penumbra. Most cells with elevated TGF-ßRI mRNA levels were microglia. TGF-ßRII co-localized with both microglial and endothelial markers while TGF-ßRIII and Alk1 were present predominantly in endothels. All four TGF-ß receptors were induced within the lesion 1 mo after the occlusion. In particular, TGF-ßRIII was further induced as compared to 72 h after MCAO. At this time point, TGF-ßRIII signal was predominantly not associated with blood vessels suggesting its microglial location. These data suggest that TGF-ß receptors are induced after MCAO in a timely and spatially regulated fashion. TGF-ß receptor expression is preceded by increased TGF-ß expression. TGF-ßRI and RII are likely to be co-expressed in microglial cells while Alk1, TGF-ßRII, and RIII in endothels within the infarct where TGF-ß1 may be their ligand. At later time points, TGF-ßRIII may also appear in glial cells to potentially affect signal transduction via TGF-ßRI and RII.

Time course, distribution and cell types of induction of transforming growth factor betas following middle cerebral artery occlusion in the rat brain.

Transforming growth factor-ßs (TGF-ß1-3) are cytokines that regulate the proliferation, differentiation, and survival of various cell types. The present study describes the induction of TGF-ß1-3 in the rat after focal ischemia at 3 h, 24 h, 72 h and 1 month after transient (1 h) or permanent (24 h) middle cerebral artery occlusion (MCAO) using in situ hybridization histochemistry and quantitative analysis. Double labeling with different markers was used to identify the localization of TGF-ß mRNA relative to the penumbra and glial scar, and the types of cells expressing TGF-ßs. TGF-ß1 expression increased 3 h after MCAO in the penumbra and was further elevated 24 h after MCAO. TGF-ß1 was present mostly in microglial cells but also in some astrocytes. By 72 h and 1 month after the occlusion, TGF-ß1 mRNA-expressing cells also appeared in microglia within the ischemic core and in the glial scar. In contrast, TGF-ß2 mRNA level was increased in neurons but not in astrocytes or microglial cells in layers II, III, and V of the ipsilateral cerebral cortex 24 h after MCAO. TGF-ß3 was not induced in cells around the penumbra. Its expression increased in only a few cells in layer II of the cerebral cortex 24 h after MCAO. The levels of TGF-ß2 and -ß3 decreased at subsequent time points. Permanent MCAO further elevated the levels of all 3 subtypes of TGF-ßs suggesting that reperfusion is not a major factor in their induction. TGF-ß1 did not co-localize with either Fos or ATF-3, while the co-localization of TGF-ß2 with Fos but not with ATF-3 suggests that cortical spreading depolarization, but not damage to neural processes, might be the mechanism of induction for TGF-ß2. The results imply that endogenous TGF-ßs are induced by different mechanisms following an ischemic attack in the brain suggesting that they are involved in distinct spatially and temporally regulated inflammatory and neuroprotective processes.

The neuroprotective functions of transforming growth factor beta proteins.

Transforming growth factor beta (TGF-ß) proteins are multifunctional cytokines whose neural functions are increasingly recognized. The machinery of TGF-ß signaling, including the serine kinase type transmembrane receptors, is present in the central nervous system. However, the 3 mammalian TGF-ß subtypes have distinct distributions in the brain suggesting different neural functions. Evidence of their involvement in the development and plasticity of the nervous system as well as their functions in peripheral organs suggested that they also exhibit neuroprotective functions. Indeed, TGF-ß expression is induced following a variety of types of brain tissue injury. The neuroprotective function of TGF-ßs is most established following brain ischemia. Damage in experimental animal models of global and focal ischemia was shown to be attenuated by TGF-ßs. In addition, support for their neuroprotective actions following trauma, sclerosis multiplex, neurodegenerative diseases, infections, and brain tumors is also accumulating. The review will also describe the potential mechanisms of neuroprotection exerted by TGF-ßs including anti-inflammatory, -apoptotic, -excitotoxic actions as well as the promotion of scar formation, angiogenesis, and neuroregeneration. The participation of these mechanisms in the neuroprotective effects of TGF-ßs during different brain lesions will also be discussed.

Distribution of mRNAs encoding transforming growth factors-beta1, -2, and -3 in the intact rat brain and after experimentally induced focal ischemia.

Transforming growth factors-beta1 (TGF-beta1), -2, and -3 form a small group of related proteins involved in the regulation of proliferation, differentiation, and survival of various cell types. Recently, TGF-betas were also demonstrated to be neuroprotective. In the present study, we investigated their distribution in the rat brain as well as their expression following middle cerebral artery occlusion. Probes were produced for all types of TGF-betas, and in situ hybridization was performed. We demonstrated high TGF-beta1 expression in cerebral cortex, hippocampus, central amygdaloid nucleus, medial preoptic area, hypothalamic paraventricular nucleus, substantia nigra, brainstem reticular formation and motoneurons, and area postrema. In contrast, TGF-beta2 was abundantly expressed in deep cortical layers, dentate gyrus, midline thalamic nuclei, posterior hypothalamic area and mamillary body, superior olive, areas of monoaminergic neurons, spinal trigeminal nucleus, dorsal vagal complex, cerebellum, and choroid plexus, and a high level of TGF-beta3 mRNA was found in cerebral cortex, hippocampus, basal amygdaloid nuclei, lateral septal nucleus, several thalamic nuclei, arcuate and supramamillary nuclei, superior colliculus, superior olive, brainstem reticular formation and motoneurons, area postrema, and inferior olive. Focal brain ischemia induced TGF-betas with markedly different expression patterns. TGF-beta1 was induced in the penumbral region of cortex and striatum, whereas TGF-beta2 and -beta3 were induced in different layers of the ipsilateral cortex. The expression of the subtypes of TGF-betas in different brain regions suggests that they are involved in the regulation of different neurons and bind to different latent TGF-beta binding proteins. Furthermore, they might have subtype-specific functions following ischemic attack.