Post-reperfusion acute MR diffusion in stroke is a potential predictor for clinical outcome in rats.

Middle cerebral artery occlusion (MCAO) models show substantial variability in outcome, introducing uncertainties in the evaluation of treatment effects. Early outcome predictors would be essential for prognostic purposes and variability control. We aimed to compare apparent diffusion coefficient (ADC) MRI data obtained during MCAO and shortly after reperfusion for their potentials in acute-phase outcome prediction. Fifty-nine male rats underwent a 45-min MCAO. Outcome was defined in three ways: 21-day survival; 24 h midline-shift and neurological scores. Animals were divided into two groups: rats surviving 21 days after MCAO (survival group, n = 46) and rats dying prematurely (non-survival/NS group, n = 13). At reperfusion, NS group showed considerably larger lesion volume and lower mean ADC of the initial lesion site (p < 0.0001), while during occlusion there were no significant group differences. At reperfusion, each survival animal showed decreased lesion volume and increased mean ADC of the initial lesion site compared to those during occlusion (p < 10-6), while NS group showed a mixed pattern. At reperfusion, lesion volume and mean ADC of the initial lesion site were significantly associated with 24 h midline-shift and neurological scores. Diffusion MRI performed soon after reperfusion has a great impact in early-phase outcome prediction, and it works better than the measurement during occlusion.

Stereolithography 3D Printing of a Heat Exchanger for Advanced Temperature Control in Wire Myography.

We report the additive manufacturing of a heat-exchange device that can be used as a cooling accessory in a wire myograph. Wire myography is used for measuring vasomotor responses in small resistance arteries; however, the commercially available devices are not capable of active cooling. Here, we critically evaluated a transparent resin material, in terms of mechanical, structural, and thermal behavior. Tensile strength tests (67.66 ± 1.31 MPa), Charpy impact strength test (20.70 ± 2.30 kJ/m2), and Shore D hardness measurements (83.0 ± 0.47) underlined the mechanical stability of the material, supported by digital microscopy, which revealed a glass-like structure. Differential scanning calorimetry with thermogravimetry analysis and thermal conductivity measurements showed heat stability until ~250 °C and effective heat insulation. The 3D-printed heat exchanger was tested in thermophysiology experiments measuring the vasomotor responses of rat tail arteries at different temperatures (13, 16, and 36 °C). The heat-exchange device was successfully used as an accessory of the wire myograph system to cool down the experimental chambers and steadily maintain the targeted temperatures. We observed temperature-dependent differences in the vasoconstriction induced by phenylephrine and KCl. In conclusion, the transparent resin material can be used in additive manufacturing of heat-exchange devices for biomedical research, such as wire myography. Our animal experiments underline the importance of temperature-dependent physiological mechanisms, which should be further studied to understand the background of the thermal changes and their consequences.

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.

CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, ß1 integrin recycling, and cell migration.

Cell attachment to the extracellular matrix (ECM) requires a balance between integrin internalization and recycling to the surface that is mediated by numerous proteins, emphasizing the complexity of these processes. Upon ligand binding in various cells, the ß1 integrin is internalized, traffics to early endosomes, and is returned to the plasma membrane through recycling endosomes. This trafficking process depends on the cyclical activation and inactivation of small guanosine triphosphatases (GTPases) by their specific guanine exchange factors (GEFs) and their GTPase-activating proteins (GAPs). In this study, we found that the cell surface antigen CD13, a multifunctional transmembrane molecule that regulates cell-cell adhesion and receptor-mediated endocytosis, also promoted cell migration and colocalized with ß1 integrin at sites of cell adhesion and at the leading edge. A lack of CD13 resulted in aberrant trafficking of internalized ß1 integrin to late endosomes and its ultimate degradation. Our data indicate that CD13 promoted ARF6 GTPase activity by positioning the ARF6-GEF EFA6 at the cell membrane. In migrating cells, a complex containing phosphorylated CD13, IQGAP1, GTP-bound (active) ARF6, and EFA6 at the leading edge promoted the ARF6 GTPase cycling and cell migration. Together, our findings uncover a role for CD13 in the fundamental cellular processes of receptor recycling, regulation of small GTPase activities, cell-ECM interactions, and cell migration.

VPAC1 receptors play a dominant role in PACAP-induced vasorelaxation in female mice.

BACKGROUND: PACAP and VIP are closely related neuropeptides with wide distribution and potent effect in the vasculature. We previously reported vasomotor activity in peripheral vasculature of male wild type (WT) and PACAP-deficient (KO) mice. However, female vascular responses are still unexplored. We hypothesized that PACAP-like activity is maintained in female PACAP KO mice and the mechanism through which it is regulated differs from that of male PACAP KO animals. METHODS: We investigated the vasomotor effects of VIP and PACAP isoforms and their selective blockers in WT and PACAP KO female mice in carotid and femoral arteries. The expression and level of different PACAP receptors in the vessels were measured by RT-PCR and Western blot. RESULTS: In both carotid and femoral arteries of WT mice, PACAP1-38, PACAP1-27 or VIP induced relaxation, without pronounced differences between them. Reduced relaxation was recorded only in the carotid arteries of KO mice as compared to their WT controls. The specific VPAC1R antagonist completely blocked the PACAP/VIP-induced relaxation in both arteries of all mice, while PAC1R antagonist affected relaxation only in their femoral arteries. CONCLUSION: In female WT mice, VPAC1 receptors appear to play a dominant role in PACAP-induced vasorelaxation both in carotid and in femoral arteries. In the PACAP KO group PAC1R activation exerts vasorelaxation in the femoral arteries but in carotid arteries there is no significant effect of the activation of this receptor. In the background of this regional difference, decreased PAC1R and increased VPAC1R availability in the carotid arteries was found.

Metformin induces significant reduction of body weight, total cholesterol and LDL levels in the elderly - A meta-analysis.

BACKGROUND: Metformin is the first-choice drug for patients with Type 2 diabetes, and this therapy is characterized by being weight neutral. However, in the elderly an additional unintentional weight loss could be considered as an adverse effect of the treatment. OBJECTIVES: We aimed to perform a meta-analysis of placebo-controlled studies investigating the body weight changes upon metformin treatment in participants older than 60 years. MATERIALS AND METHODS: PubMed, EMBASE and the Cochrane Library were searched. We included at least 12 week-long studies with placebo control where the mean age of the metformin-treated patients was 60 years or older and the body weight changes of the patients were reported. We registered our protocol on PROSPERO (CRD42017055287). RESULTS: From the 971 articles identified by the search, 6 randomized placebo-controlled studies (RCTs) were included in the meta-analysis (n = 1541 participants). A raw difference of -2.23 kg (95% CI: -2.84 --1.62 kg) body weight change was detected in the metformin-treated groups as compared with that of the placebo groups (p<0.001). Both total cholesterol (-0.184 mmol/L, p<0.001) and LDL cholesterol levels (-0.182 mmol/L, p<0.001) decreased upon metformin-treatment. CONCLUSIONS: Our meta-analysis of RCTs showed a small reduction of body weight together with slight improvement of the blood lipid profile in patients over 60 years. With regard to the risk of unintentional weight loss, metformin seems to be a safe agent in the population of over 60 years. Our results also suggest that metformin treatment may reduce the risk of major coronary events (-4-5%) and all-cause mortality (-2%) in elderly diabetic populations.

Pituitary adenylate cyclase-activating polypeptide ameliorates vascular dysfunction induced by hyperglycaemia.

BACKGROUND: Short-lasting hyperglycaemia occurs frequently in prediabetes and poorly controlled diabetes mellitus leading to vascular damage. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to play a protective role in vascular complications of diabetes; moreover, antioxidant effects of PACAP were also described. Therefore, we hypothesized that PACAP exerts protective effects in short-term hyperglycaemia-induced vascular dysfunctions. METHODS: After short-term hyperglycaemia, acetylcholine-induced and sodium nitroprusside-induced vascular relaxation of mouse carotid arteries were tested with a myograph with or without the presence of PACAP or superoxide dismutase. Potential direct antioxidant superoxide-scavenging action of pituitary adenylate cyclase-activating peptide was tested with pyrogallol autoxidation assay; furthermore, the effect of pituitary adenylate cyclase-activating peptide or superoxide dismutase was investigated on hyperglycaemia-associated vascular markers. RESULTS: PACAP administration resulted in reduced endothelial dysfunction after a 1-h hyperglycaemic episode. PACAP was able to restore acetylcholine-induced relaxation of the vessels and improved sodium nitroprusside-induced relaxation. This effect was comparable to the protective effect of superoxide dismutase, but PACAP was unable to directly scavenge superoxide produced by autoxidation of pyrogallol. Endothelial dysfunction was associated with elevated levels of fibroblast growth factor basic, matrix metalloproteinase 9 and nephroblastoma overexpressed gene proteins. Their release was reduced by PACAP administration. CONCLUSION: These results suggest a strong protective role of PACAP in the vascular complications of diabetes.

Aging-Induced Modulation of Pituitary Adenylate Cyclase-Activating Peptide- and Vasoactive Intestinal Peptide-Induced Vasomotor Responses in the Arteries of Mice.

Pituitary adenylate cyclase-activating peptide (PACAP; 1-38 and 1-27) and vasoactive intestinal peptide (VIP) are related neuropeptides of the secretin/glucagon family. Overlapping signaling through G-protein-coupled receptors mediates their vasomotor activity. We previously showed that PACAP deficiency (PACAP-KO) shifts the mechanisms of vascular response and maintains arterial relaxation through the VIP backup mechanism and (mainly) its VPAC1R, but their age-dependent modulation is still unknown. We hypothesized that backup mechanisms exist, which maintain the vasomotor activity of these peptides also in older age. Thus, we investigated the effects of exogenous VIP and PACAP peptides in isolated carotid arteries of 2- and 15-month-old wild-type (WT) and PACAP-KO mice. All peptides induced relaxation in the arteries of young WT mice, whereas in young PACAP-KO mice PACAP1-27 and VIP, but not PACAP1-38, induced relaxation. Unlike VIP, PACAP-induced vasomotor responses were reduced in aging WT mice. However, in the arteries of aging PACAP-KO mice, PACAP1-27- and VIP-induced responses were reduced, but PACAP1-38 showed a greater vasomotor response compared to that of young PACAP-KO animals. There were no significant differences between the vasomotor responses of aging WT and PACAP-KO mice. Our data suggest that, in the absence of PACAP both in young and old ages, the vascular response is mediated through backup mechanisms, most likely VIP, maintaining proper vascular relaxation in aging-induced PACAP insufficiency.

Backup Mechanisms Maintain PACAP/VIP-Induced Arterial Relaxations in Pituitary Adenylate Cyclase-Activating Polypeptide-Deficient Mice.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide in the VIP/secretin/glucagon peptide superfamily. Two active forms, PACAP1-38 and PACAP1-27, act through G protein-coupled receptors, the PAC1 and VPAC1/2 receptors. Effects of PACAP include potent vasomotor activity. Vasomotor activity and organ-specific vasomotor effects of PACAP-deficient mice have not yet been investigated; thus, the assessment of its physiological importance in vasomotor functions is still missing. We hypothesized that backup mechanisms exist to maintain PACAP pathway activity in PACAP knockout (KO) mice. Thus, we investigated the vasomotor effects of exogenous vasoactive intestinal peptide (VIP) and PACAP polypeptides in PACAP wild-type (WT) and PACAP-deficient (KO) male mice. METHODS: Carotid and femoral arteries were isolated from 8- to 12-week-old male WT and PACAP-KO mice. Vasomotor responses were measured with isometric myography. RESULTS: In the arteries of WT mice the peptides induced relaxations, which were significantly greater to PACAP1-38 than to PACAP1-27 and VIP. In KO mice, PACAP1-38 did not elicit relaxation, whereas PACAP1-27 and VIP elicited significantly greater relaxation in KO mice than in WT mice. The specific PAC1R and VPAC1R antagonist completely blocked the PACAP-induced relaxations. CONCLUSION: Our data suggest that in PACAP deficiency, backup mechanisms maintain arterial relaxations to polypeptides, indicating an important physiological role for the PACAP pathway in the regulation of vascular tone.

Hypertension-Induced Enhanced Myogenic Constriction of Cerebral Arteries Is Preserved after Traumatic Brain Injury.

Traumatic brain injury (TBI) was shown to impair pressure-induced myogenic response of cerebral arteries, which is associated with vascular and neural dysfunction and increased mortality of TBI patients. Hypertension was shown to enhance myogenic tone of cerebral arteries via increased vascular production of 20-hydroxyeicosatrienoic acid (HETE). This adaptive mechanism protects brain tissue from pressure/volume overload; however, it can also lead to increased susceptibility to cerebral ischemia. Although both effects may potentiate the detrimental vascular consequences of TBI, it is not known how hypertension modulates the effect of TBI on myogenic responses of cerebral vessels. We hypothesized that in hypertensive rats, the enhanced myogenic cerebrovascular response is preserved after TBI. Therefore, we investigated the myogenic responses of isolated middle cerebral arteries (MCA) of normotensive and spontaneously hypertensive rats (SHR) after severe impact acceleration diffuse brain injury. TBI diminished myogenic constriction of MCAs isolated from normotensive rats, whereas the 20-HETE-mediated enhanced myogenic response of MCAs isolated from SHRs was not affected by TBI. These results suggest that the optimal cerebral perfusion pressure values and vascular signaling pathways can be different and, therefore, should be targeted differently in normotensive and hypertensive patients following TBI.

Hemolyzed Blood Elicits a Calcium Antagonist and High CO2 Reversible Constriction via Elevation of [Ca2+]i in Isolated Cerebral Arteries.

During acute subarachnoid hemorrhage, blood is hemolyzed, which is followed by a significant cerebrovascular spasm resulting in a serious clinical condition. Interestingly, however, the direct vasomotor effect of perivascular hemolyzed blood (HB) has not yet been characterized, preventing the assessment of contribution of vasoconstrictor mechanisms deriving from brain tissue and/or blood and development of possible treatments. We hypothesized that perivascular HB reduces the diameter of the cerebral arteries (i.e., basilar artery [BA]; middle cerebral artery [MCA]) by elevating vascular tissue [Ca2+]i level. Vasomotor responses were measured by videomicroscopy and intracellular Ca2+ by the Fura2-AM ratiometric method. Adding HB to the vessel chamber reduced the diameter significantly (BA: from 264 ± 7 to 164 ± 11 µm; MCA: from 185 ± 15 to 155 ± 14 µm), which was reversed to control level by wash-out of HB. Potassium chloride (KCl), HB, serum, hemolyzed red blood cell (RBC), plasma, and platelet suspension (PLTs) elicited significant constrictions of isolated basilar arteries. There was a significant increase in K+ concentration in hemolyzed HB (7.02 ± 0.22 mmol/L) compared to Krebs' solution (6.20 ± 0.01 mmol/L). Before HB, acetylcholine (ACh), sodium-nitroprussid (SNP), nifedipin, and CO2 elicited substantial dilations in cerebral arteries. In contrast, in the presence of HB dilations to ACh, SNP decreased, but not to nifedipine and CO2. After washout of HB, nitric oxide-mediated dilations remained significantly reduced compared to control. HB significantly increased the ratiometric Ca signal, which returned to control level after washout. In conclusion, perivascular hemolyzed blood elicits significant-nifedipine and high CO2 reversible-constrictions of isolated BAs and MCAs, primarily by increasing intracellular Ca2+, findings that can contribute to the refinement of local treatment of subarachnoid hemorrhage.

From Newborn to Senescence Morphological and Functional Remodeling Leads to Increased Contractile Capacity of Arteries.

Aging induces substantial morphological and functional changes in vessels. We hypothesized that due to morphological remodeling the total contractile forces of arteries increase, especially in older age as a function of age. Mean arterial blood pressure of rats and morphological and functional characteristics of isolated carotid arteries rats, from newborn to senescent, were assessed. The arterial blood pressure of rats increased significantly from 0.25 to the age of 6 months, and then it reached a level, which was maintained until age of 30 months. Wall lumen and wall thickness increased with age, mostly due to media (smooth muscle) thickening, whereas wall tension gradually reduced with age. Contractions of arteries to nonreceptor-mediated vasomotor agent (KCl, 60mM) increased in three consecutive age groups, whereas contractility first increased (until 2 months), then it did not change further with aging. Norepinephrine-induced contractions initially increased in young age and then did not change further in older age. These findings suggest that during normal aging due to remodeling of arterial wall (smooth muscle) the contractile capacity of arteries increases, which seems to be independent from systemic blood pressure. Thus, arterial remodeling can favor the development of increased circulatory resistance in older age.

Transient Receptor Potential Vanilloid-1 Channels Contribute to the Regulation of Acid- and Base-Induced Vasomotor Responses.

pH changes can influence local blood flow, but the mechanisms of how acids and bases affect vascular tone is not fully clarified. Transient receptor potential vanilloid-1 (TRPV1) channels are expressed in vessels and can be activated by pH alterations. Thus, we hypothesized that TRPV1 channels are involved in the mediation of vascular responses to acid-base changes. Vasomotor responses to HCl, NaOH, and capsaicin were measured in isolated murine carotid and tail skin arteries. The function of TRPV1 was blocked by either of three approaches: Trpv1 gene disruption, pharmacological blockade with a TRPV1 antagonist (BCTC), and functional impairment of mainly neural TRPV1 channels (desensitization). In each artery type of control mice, HCl caused relaxation but NaOH contraction, and both responses were augmented after genetic or pharmacological TRPV1 blockade. In arteries of TRPV1-desensitized mice, HCl-induced relaxation did not differ from controls, whereas NaOH-induced contraction was augmented. All three types of TRPV1 blockade had more pronounced effects in carotid than in tail skin arteries. We conclude that TRPV1 channels limit the vasomotor responses to changes in pH. While base-induced arterial contraction is regulated primarily by neural TRPV1 channels, acid-induced arterial relaxation is modulated by TRPV1 channels located on nonneural vascular structures.

The Beta-1-Receptor Blocker Nebivolol Elicits Dilation of Cerebral Arteries by Reducing Smooth Muscle [Ca2+]i.

RATIONALE: Nebivolol is known to have beta-1 blocker activity, but it was also suggested that it elicits relaxation of the peripheral arteries in part via release of nitric oxide (NO). However, the effect of nebivolol on the vasomotor tone of cerebral arteries is still unclear. OBJECTIVE: To assess the effects of nebivolol on the diameter of isolated rat basilar arteries (BA) in control, in the presence of inhibitors of vasomotor signaling pathways of know action and hemolysed blood. METHODS AND RESULTS: Vasomotor responses were measured by videomicroscopy and the intracellular Ca2+ by the Fura-2 AM ratiometric method. Under control conditions, nebivolol elicited a substantial dilation of the BA (from 216±22 to 394±20 µm; p<0.05) in a concentration-dependent manner (10-7 to 10-4 M). The dilatation was significantly reduced by endothelium denudation or by L-NAME (inhibitor of NO synthase) or by SQ22536 (adenylyl cyclase blocker). Dilatation of BA was also affected by beta-2 receptor blockade with butoxamine, but not by the guanylate cyclase blocker ODQ. Interestingly, beta-1 blockade by atenolol inhibited nebivolol-induced dilation. Also, the BKCa channel blocker iberiotoxin and KCa channel inhibitor TEA significantly reduced nebivolol-induced dilation. Nebivolol significantly reduced smooth muscle Ca2+ level, which correlated with the increases in diameters and moreover it reversed the hemolysed blood-induced constriction of BA. CONCLUSIONS: Nebivolol seems to have an important dilator effect in cerebral arteries, which is mediated via several vasomotor mechanisms, converging on the reduction of smooth muscle Ca2+ levels. As such, nebivolol may be effective to improve cerebral circulation in various diseased conditions, such as hemorrhage.

Pituitary adenylate cyclase-activating polypeptide (PACAP) induces relaxations of peripheral and cerebral arteries, which are differentially impaired by aging.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a well-known neuropeptide, which also has vasomotor effects. However, little is known regarding its age-related and organ-specific vasomotor effects. We hypothesized that the vasomotor effects of PACAP depend on the tissue origin of the vessels and aging substantially modulates its actions. Thus, carotid (CA) and basilar arteries (BA) were isolated from young (2 months old), middle age (12 months old), and old (30 months old) rats. Their vasomotor responses were measured with an isometric myograph (DMT610M) in response to cumulative concentrations of PACAP1-38 (10(-9)-10(-6) M). PACAP1-38 induced (1) significantly greater concentration-dependent relaxations in CA compared to that of BA of young, middle age, and old rats; (2) relaxations of CA significantly decreased, whereas they did not change substantially in BA, as a function of age; (3) sodium nitroprusside (SNP)-induced relaxation did not change after PACAP1-38 administration in any conditions; and (4) inhibition of PAC1 receptors by selective PAC1 receptor blocker (PACAP6-38) completely diminished the responses to PACAP in all age groups of BA and CA. In conclusion, these findings suggest that PACAP1-38 has greater vasomotor effect in CA than that in BA, whereas aging has less effect on PACAP-induced relaxation of cerebral arteries and BA than that in peripheral arteries and CA suggesting that the relaxation to PACAP is maintained in cerebral arteries even in old age.

Age determines the magnitudes of angiotensin II-induced contractions, mRNA, and protein expression of angiotensin type 1 receptors in rat carotid arteries.

In this study, we hypothesized that aging alters angiotensin II (Ang II)-induced vasomotor responses and expression of vascular mRNA and protein angiotensin type 1 receptor (AT1R). Thus, carotid arteries were isolated from the following age groups of rats: 8 days, 2-9 months, 12-20 months, and 20-30 months, and their vasomotor responses were measured in a myograph after repeated administrations of Ang II. Vascular relative AT1R mRNA level was determined by quantitative reverse-transcriptase polymerase chain reaction and the AT1R protein density was measured by Western blot. Contractions to the first administration of Ang II increased from 8 days to 6 months and then they decreased to 30 months. In general, second administration of Ang II elicited reduced contractions, but they also increased from 8 days until 2 months and then they decreased to 30 months. Similarly the AT1R mRNA level increased from 8 days to 12 months and then decreased to 30 months. Similarly the AT1R protein density increased from 8 days until 16 months and then they decreased to 30 months. The pattern of these changes correlated with functional vasomotor data. We conclude that aging (newborn to senescence) has substantial effects on Ang II-induced vasomotor responses and AT1R signaling suggesting the importance of genetic programs.