Pannexins are capable of forming in the cell membrane anion channels with relatively low conductivity, as well as channels with high nonspecific conductivity that can transport from the cytoplasm into the extracellular space not only inorganic ions but also low molecular weight metabolites with a molecular weight below 1 kDa, in particular ATP, glutamate, glutathione and others. Due to substantial differences in specificity and conductivity, pannexins are involved both in the normal physiological regulation of body functions and in a variety of pathological processes. The review examines the role of pannexin 1 (Panx1) in the occurrence and progression of pathologies of the central nervous system such as ischemia, Parkinson’s and Alzheimer’s diseases, and neuropathic pain. Blockade of Panx1 diminishes the negative consequences of pathological processes, indicating that Panx1 channels may be a promising therapeutic target for the pharmacological correction of brain disorders.

Diabetes mellitus is associated with various complications, mainly caused by the chronic exposure of the cells to high glucose (HG) concentrations. The effects of long-term HG exposure in vitro accompanied by lipopolysaccharide (LPS) application on astrocytes are relatively unknown. We used cell medium with normal (NG, 5.5 mM) or high glucose (HG, 25 mM) for rat astrocyte cultures and measured the release of NO, IL-6, β-hexosaminidase and cell survival in response to LPS. We first demonstrated that HG long-term incubation of astrocytes increased the release of β-hexosaminidase without decreasing MTT-detected cell survival, suggesting that there is no cell membrane damage or astrocyte death but could be lysosome exocytosis. Different from what was observed for NG, all LPS concentrations tested at HG resulted in an increase in IL-6, and this was detected for both 6 h and 48 h treatments. Interestingly, β-hexosaminidase level increased after 48 h of LPS and only at HG. The NO release from astrocytes also increased with LPS application at HG but was less significant. These data endorsed the original hypothesis that long-term hyperglycemia increases proinflammatory activation of astrocytes, and β-hexosaminidase could be a specific marker of excessive activation of astrocytes associated with exocytosis.

Protease-activated receptors (PARs) are involved not only in hemostasis but also in the development of ischemic brain injury. In the present work, we examined in vivo effects of a new peptide (AP9) composing Asn47-Phen55 of PAR1 "tethered ligand" generated by activated protein C. We chose a mouse model of photothrombosis (PT)-induced ischemia to assess AP9 effects in vivo. To reveal the molecular mechanism of AP9 action, mice lacking β-arrestin-2 were used. AP9 was injected intravenously once 10 min before PT at doses of 0.2, 2, or 20 mg/kg, or twice, that is, 10 min before and 1 h after PT at a dose of 20 mg/kg. Lesion volume was measured by magnetic resonance imaging and staining of brain sections with tetrazolium salt. Neurologic deficit was estimated using the cylinder and the grid-walk tests. Blood-brain barrier (BBB) disruption was assessed by Evans blue dye extraction. Eosin-hematoxylin staining and immunohistochemical staining were applied to evaluate the number of undamaged neurons and activated glial cells in the penumbra. A single administration of AP9 (20 mg/kg), as well as its two injections (20 mg/kg), decreased brain lesion volume. A double administration of AP9 also reduced BBB disruption and neurological deficit in mice. We did not observe the protective effect of AP9 in mice lacking β-arrestin-2 after PT. Thus, we demonstrated for the first time protective properties of a PAR1 agonist peptide, AP9, in vivo. β-Arrestin-2 was required for the protective action of AP9 in PT-induced brain ischemia.

Thrombin is a multifunctional serine protease that attracts the attention of many researchers in particular in connection with a wide range of its effects in the nervous tissue. It is known that the main thrombin receptor is a protease-activated type 1 receptor, the functional activity of which is associated with adapter protein β-arrestin-2. Here we assessed potential involvement of β-arrestin-2 in the thrombin toxicity both in vitro and in vivo using gene knockout mice. It was found that thrombin induced dose-dependent cell death of cultured β-arrestin-2–/– astrocytes 48 h after the exposure. In contrast, thrombin did not exert any effects on the survival of astrocytes from wild-type animals. The in vivo study showed that β-arrestin-2 gene knockout did not alter the severity of the focal photoinduced cerebral ischemia aftereffects, which could involve additional cell types and molecular mechanisms in nervous tissue damage. Our findings demonstrate for the first time the necessity of β-arrestin-2 for the survival of mouse astrocytes under the toxic action of thrombin. At the CNS level, however, further studies are required to determine the key targets of this protease in each cell types of the nervous tissue and to clarify the role of β-arrestin signaling in neuroprotection.

Background: Photothrombosis is a minimally invasive method for induction of cortical ischemia. However, different ways of applying some methods to assess photothrombosis-induced damage need to be developed. New Methods: We applied the tongue protrusion test and H&E staining of brain sections to detect ischemic damage after photothrombosis. Evaluation of the local status of the BBB using Evans blue dye was proposed. We also assessed the sensitivity of the grid-walk test. Moreover, we examined the interchangeability of MRI and TTC staining to measure lesion volume. Results: We evaluated ischemic outcomes at 24 h after photothrombosis in mice. The tongue protrusion test did not reveal impairments of the neurological status whereas the grid-walk test showed the high sensitivity. Using histological techniques, we determined the reduction in the number of neurons with normal morphology in the penumbra. 3D reconstruction of the brain, which reflected Evans blue dye distribution in the nervous tissue, revealed BBB disruption in areas remote from the ischemic core. We also showed the strong correlation between damage volumes assessed by MRI and TTC staining. Comparison with Existing Methods: The present work demonstrates the efficacy of the classical histological approach and TTC staining that are more affordable than MRI and immunohistochemical methods. Detection of 3D distribution of Evans blue dye in the brain in contrast to its total extraction reveals BBB damage in details. Conclusions: We proposed the simple methods for describing the severity of brain ischemia at the cellular and whole organism levels without significant labor and financial expenditures.