NO Deficiency Compromises Inter- and Intrahemispheric Blood Flow Adaptation to Unilateral Carotid Artery Occlusion.

Carotid artery stenosis (CAS) affects approximately 5-7.5% of older adults and is recognized as a significant risk factor for vascular cognitive impairment (VCI). The impact of CAS on cerebral blood flow (CBF) within the ipsilateral hemisphere relies on the adaptive capabilities of the cerebral microcirculation. In this study, we aimed to test the hypothesis that the impaired availability of nitric oxide (NO) compromises CBF homeostasis after unilateral carotid artery occlusion (CAO). To investigate this, three mouse models exhibiting compromised production of NO were tested: NOS1 knockout, NOS1/3 double knockout, and mice treated with the NO synthesis inhibitor L-NAME. Regional CBF changes following CAO were evaluated using laser-speckle contrast imaging (LSCI). Our findings demonstrated that NOS1 knockout, NOS1/3 double knockout, and L-NAME-treated mice exhibited impaired CBF adaptation to CAO. Furthermore, genetic deficiency of one or two NO synthase isoforms increased the tortuosity of pial collaterals connecting the frontoparietal and temporal regions. In conclusion, our study highlights the significant contribution of NO production to the functional adaptation of cerebrocortical microcirculation to unilateral CAO. We propose that impaired bioavailability of NO contributes to the impaired CBF homeostasis by altering inter- and intrahemispheric blood flow redistribution after unilateral disruption of carotid artery flow.

Blood oxygen regulation via P2Y12R expressed in the carotid body.

BACKGROUND: Peripheral blood oxygen monitoring via chemoreceptors in the carotid body (CB) is an integral function of the autonomic cardiorespiratory regulation. The presence of the purinergic P2Y12 receptor (P2Y12R) has been implicated in CB; however, the exact role of the receptor in O2 sensing and signal transduction is unknown. METHODS: The presence of P2Y12R was established by immunoblotting, RT qPCR and immunohistochemistry. Primary glomus cells were used to assess P2Y12R function during hypoxia and hypercapnia, where monoamines were measured by HPLC; calcium signal was recorded utilizing OGB-1 and N-STORM Super-Resolution System. Ingravescent hypoxia model was tested in anaesthetized mice of mixed gender and cardiorespiratory parameters were recorded in control and receptor-deficient or drug-treated experimental animals. RESULTS: Initially, the expression of P2Y12R in adult murine CB was confirmed. Hypoxia induced a P2Y12R-dependent release of monoamine transmitters from isolated CB cells. Receptor activation with the endogenous ligand ADP promoted release of neurotransmitters under normoxic conditions, while blockade disrupted the amplitude and duration of the intracellular calcium concentration. In anaesthetised mice, blockade of P2Y12R expressed in the CB abrogated the initiation of compensatory cardiorespiratory changes in hypoxic environment, while centrally inhibited receptors (i.e. microglial receptors) or receptor-deficiency induced by platelet depletion had limited influence on the physiological adjustment to hypoxia. CONCLUSIONS: Peripheral P2Y12R inhibition interfere with the complex mechanisms of acute oxygen sensing by influencing the calcium signalling and the release of neurotransmitter molecules to evoke compensatory response to hypoxia. Prospectively, the irreversible blockade of glomic receptors by anti-platelet drugs targeting P2Y12Rs, propose a potential, formerly unrecognized side-effect to anti-platelet medications in patients with pulmonary morbidities.

Disruption of Vitamin D Signaling Impairs Adaptation of Cerebrocortical Microcirculation to Carotid Artery Occlusion in Hyperandrogenic Female Mice.

Vitamin D deficiency contributes to the pathogenesis of age-related cerebrovascular diseases, including ischemic stroke. Sex hormonal status may also influence the prevalence of these disorders, indicated by a heightened vulnerability among postmenopausal and hyperandrogenic women. To investigate the potential interaction between sex steroids and disrupted vitamin D signaling in the cerebral microcirculation, we examined the cerebrovascular adaptation to unilateral carotid artery occlusion (CAO) in intact, ovariectomized, and hyperandrogenic female mice with normal or functionally inactive vitamin D receptor (VDR). We also analyzed the morphology of leptomeningeal anastomoses, which play a significant role in the compensation. Ablation of VDR by itself did not impact the cerebrocortical adaptation to CAO despite the reduced number of pial collaterals. While ovariectomy did not undermine compensatory mechanisms following CAO, androgen excess combined with VDR inactivity resulted in prolonged hypoperfusion in the cerebral cortex ipsilateral to the occlusion. These findings suggest that the cerebrovascular consequences of disrupted VDR signaling are less pronounced in females, providing a level of protection even after ovariectomy. Conversely, even short-term androgen excess with lacking VDR signaling may lead to unfavorable outcomes of ischemic stroke, highlighting the complex interplay between sex steroids and vitamin D in terms of cerebrovascular diseases.

Application of Injectable, Crosslinked, Fibrin-Containing Hyaluronic Acid Scaffolds for In Vivo Remodeling.

The present research aimed to characterize soft tissue implants that were prepared with the use of crosslinked hyaluronic acid (HA) using two different crosslinkers and multiple reagent concentrations, alone or in combination with fibrin. The effect of the implants was evaluated in an in vivo mouse model, after 4 weeks in one group and after 12 weeks in the other. The explants were compared using analytical methods, evaluating microscopic images, and a histology analysis. The kinetics of the degradation and remodeling of explants were found to be greatly dependent on the concentration and type of crosslinker; generally, divinyl sulfone (DVS) resists degradation more effectively compared to butanediol diglycidyl ether (BDDE). The presence of fibrin enhances the formation of blood vessels, and the infiltration of cells and extracellular matrix. In summary, if the aim is to create a soft tissue implant with easier degradation of the HA content, then the use of 2-5% BDDE is found to be optimal. For a longer degradation time, 5% DVS is the more suitable crosslinker. The use of fibrin was found to support the biological process of remodeling, while keeping the advances of HA in void filling, enabling the parallel degradation and remodeling processes.

The Hypertensive Effect of Amphotericin B-Containing Liposomes (Abelcet) in Mice: Dissecting the Roles of C3a and C5a Anaphylatoxins, Macrophages and Thromboxane.

Liposomal amphotericin B (Abelcet) can cause infusion (anaphylactoid) reactions in patients whose mechanism is poorly understood. Here, we used mice to investigate the role of complement (C) receptors and the cellular sources of vasoactive mediators in these reactions. Anesthetized male NMRI and thromboxane prostanoid receptor (TP) or cyclooxygenase-1 (COX-1)-deficient and wild type C57Bl6/N mice were intravenously injected with Abelcet at 30 mg/kg. Mean arterial blood pressure (MABP) and heart rate (HR) were measured. In untreated mice, Abelcet caused a short (15 min) but large (30%) increase in MABP. C depletion with cobra venom factor (CVF) and inhibition of C5a receptors with DF2593A considerably prolonged, while C3aR inhibition with SB290157 significantly decreased the hypertensive effect. Likewise, the hypertensive response was abolished in COX-1- and TP-deficient mice. CVF caused a late hypertension in TP-deficient mice. Both macrophage depletion with liposomal clodronate and blockade of platelet GPIIb/IIIa receptors with eptifibatide prolonged the hypertensive effect. The early phase of the hypertensive effect is COX-1- and TP-receptor-dependent, partly mediated by C3aR. In contrast, the late phase is under the control of vasoactive mediators released from platelets and macrophages subsequent to complement activation and C5a binding to its receptor.

A Minimally Invasive, Fast Spinal Cord Lateral Hemisection Technique for Modeling Open Spinal Cord Injuries in Rats.

Open spinal cord injury techniques modeling laceration-like injuries are time-consuming and invasive because they involve laminectomy. This new technique eliminates laminectomy by removing two spinous processes and lifting, then tilting the caudal vertebral arch. The surgical area opens up without the need for laminectomy. Lateral hemisection is then performed with direct visible control under a microscope. The trauma is minimized, requiring only a small bone wound. This technique has several advantages: it is faster and, therefore, less of a burden for the animal, and the bone wound is smaller. Because the laminectomy is eliminated, there is less chance for unwanted injury to the spinal cord, and there are no bone splinters that can cause problems (bone splinters embedded in the spinal cord can cause swelling and secondary damage). The vertebral canal remains intact. The main limitation is that the hemisection can only be performed in the intervertebral spaces. The results show that this technique can be performed much faster than the traditional surgical approach, using laminectomy (11 min vs. 35 min). This technique can be useful for researchers working with animal models of open spinal cord injury as it is widely adaptable and does not require any additional specialized instrumentation.

Microglia modulate blood flow, neurovascular coupling, and hypoperfusion via purinergic actions.

Microglia, the main immunocompetent cells of the brain, regulate neuronal function, but their contribution to cerebral blood flow (CBF) regulation has remained elusive. Here, we identify microglia as important modulators of CBF both under physiological conditions and during hypoperfusion. Microglia establish direct, dynamic purinergic contacts with cells in the neurovascular unit that shape CBF in both mice and humans. Surprisingly, the absence of microglia or blockade of microglial P2Y12 receptor (P2Y12R) substantially impairs neurovascular coupling in mice, which is reiterated by chemogenetically induced microglial dysfunction associated with impaired ATP sensitivity. Hypercapnia induces rapid microglial calcium changes, P2Y12R-mediated formation of perivascular phylopodia, and microglial adenosine production, while depletion of microglia reduces brain pH and impairs hypercapnia-induced vasodilation. Microglial actions modulate vascular cyclic GMP levels but are partially independent of nitric oxide. Finally, microglial dysfunction markedly impairs P2Y12R-mediated cerebrovascular adaptation to common carotid artery occlusion resulting in hypoperfusion. Thus, our data reveal a previously unrecognized role for microglia in CBF regulation, with broad implications for common neurological diseases.

Involvement of P2Y12 receptors in a nitroglycerin-induced model of migraine in male mice.

BACKGROUND AND PURPOSE: P2Y12 receptors regulate different forms of pain and inflammation. In this study, we investigated the participation of P2Y12 receptors in an animal model of migraine. EXPERIMENTAL APPROACH: We tested the effect of the centrally administered selective P2Y12 antagonist PSB-0739 and P2Y12 receptor gene (P2ry12-/- ) deficiency in acute nitroglycerin-treated mice. Additionally, platelet depletion was used to investigate the role of platelet P2Y12 receptors during migraine-like pain. KEY RESULTS: Nitroglycerin induced sensory hypersensitivity of C57BL/6 wild-type (P2ry12+/+ ) mice accompanied by an increase in c-fos and CGRP expression in the upper cervical spinal cord (C1-C2) and trigeminal nucleus caudalis. Similar changes were also observed in P2Y12 gene-deficient (P2ry12-/- ) mice. Prophylactic intrathecal application of PSB-0739 reversed thermal hyperalgesia and head grooming time in wild-type mice but had no effect in P2ry12-/- mice. Furthermore, PSB-0739 was also effective when applied as a post-treatment. PSB-0739 administration suppressed the expression of c-fos in C1-C2 and trigeminal nucleus caudalis, and decreased the levels of dopamine and 5-hydroxytryptamine in C1-C2 in wild-type mice. Nitroglycerin treatment itself did not change adenosine diphosphate (ADP)-induced platelet activation measured by CD62P up-regulation in wild-type mice. Platelet depletion by anti-mouse CD41 antibody and clopidogrel attenuated nitroglycerin-induced thermal hypersensitivity and head grooming time in mice. CONCLUSION AND IMPLICATIONS: Our findings show that acute inhibition of P2Y12 receptors alleviates migraine-like pain in mice by modulating the expression of c-fos and that platelet P2Y12 receptors might contribute to this effect. Thus the blockade of P2Y12 receptors may have therapeutic potential against migraine.

Crosslinked Hyaluronic Acid Gels with Blood-Derived Protein Components for Soft Tissue Regeneration.

Hyaluronic acid (HA) is an ideal initial material for preparing hydrogels, which may be used as scaffolds in soft tissue engineering based on their advantageous physical and biological properties. In this study, two crosslinking agents, divinyl sulfone (DVS) and butanediol diglycidyl ether, were used to investigate their effect on the properties of HA hydrogels. As HA hydrogels alone do not promote cell adhesion on the scaffold, fibrin and serum from platelet-rich fibrin (SPRF) were combined with the scaffold; the aim was to create a material intended to be used as soft tissue implant that facilitates new tissue formation, and degrades over time. The chemical changes were characterized and cell attachment capacity of the protein-containing gels was examined using human mesenchymal stem cells, and viability was assessed using live-dead staining. Fourier-transform infrared measurements revealed that linking fibrin into the gel was more effective than linking SPRF. The scaffolds were found to be able to support cell adherence onto the hydrogels, and the best result was achieved when HA was crosslinked with DVS and contained fibrin. The most promising derivative, 5% DVS-crosslinked fibrin-containing hydrogel, was injected subcutaneously into C57BL/6 mice for 12 weeks. The scaffold was proven to be biocompatible, remodeling, and vascularization occurred, while shape and integrity were maintained. Impact statement Fibrin was combined with crosslinked hyaluronic acid (HA) for regenerative application, the structure of the combination of crosslinked HA with blood-derived protein was analyzed and effective coating was proven. It was observed that the fibrin content led to better mesenchymal stem cell attachment in vitro. The compositions showed biocompatibility, connective tissue and vascularization took place when implanted in vivo. Thus, a biocompatible, injectable gel was produced, which is a potential candidate for soft tissue implantation.

Ablation of Vitamin D Signaling Compromises Cerebrovascular Adaptation to Carotid Artery Occlusion in Mice.

Vitamin D insufficiency has been associated with increased incidence and severity of cerebrovascular disorders. We analyzed the impact of impaired vitamin D signaling on the anatomical and functional aspects of cerebrovascular adaptation to unilateral carotid artery occlusion (CAO), a common consequence of atherosclerosis and cause of ischemic stroke. Cerebrocortical blood flow (CoBF) showed a significantly increased drop and delayed recovery after CAO in mice carrying a functionally inactive vitamin D receptor (VDR) with the most sustained perfusion deficit in the temporal cortex. To identify the cause(s) for this altered adaptation, the extent of compensatory blood flow increase in the contralateral carotid artery and the morphology of pial collaterals between the anterior and middle cerebral arteries were determined. Whereas VDR deficiency had no significant influence on the contralateral carotid arterial blood flow increase, it was associated with decreased number and increased tortuosity of pial anastomoses resulting in unfavorable changes of the intracranial collateral circulation. These results indicate that VDR deficiency compromises the cerebrovascular adaptation to CAO with the most sustained consequences in the temporal cortex. The dysregulation can be attributed to the altered development and function of pial collateral circulation whereas extracranial vessels may not be impaired.

CB1 receptor-mediated respiratory depression by endocannabinoids.

Endocannabinoids (ECs) are bioactive lipid mediators acting on two distinct cannabinoid receptors (CB1 and CB2), which are ubiquitously expressed in many tissues including the respiratory system. Despite numerous experimental data showing that cannabinomimetics influence respiration, the role of endogenously produced ECs in respiratory control has not been verified yet. Pulse oximetry was used in the present study to directly measure changes in respiratory parameters during elevation of EC levels. The cannabinoid reuptake inhibitor AM-404 (10mgkg-1, i.v.), but not its vehicle, induced a transient reduction of respiratory rate with a concomitant depression of arterial oxygen saturation and increase in breath distension in wild-type mice. In contrast, CB1 knock-out mice showed no alteration in any of these parameters upon administration of AM-404. Our results imply that the EC system has an important role in the physiological control of respiration by modulating the respiratory rate and consequently influencing arterial oxygen saturation. Furthermore, this mechanism is entirely dependent on CB1 receptors.

Evaluation of Laser Speckle Contrast Imaging for the Assessment of Oral Mucosal Blood Flow following Periodontal Plastic Surgery: An Exploratory Study.

The laser speckle contrast imaging (LSCI) is proved to be a reliable tool in flap monitoring in general surgery; however, it has not been evaluated in oral surgery yet. We applied the LSCI to compare the effect of a xenogeneic collagen matrix (Geistlich Mucograft®) to connective tissue grafts (CTG) on the microcirculation of the modified coronally advanced tunnel technique (MCAT) for gingival recession coverage. Gingival microcirculation and wound fluid were measured before and after surgery for six months at twenty-seven treated teeth. In males, the flap microcirculation was restored within 3 days for both grafts followed by a hyperemic response. During the first 8 days the blood flow was higher at xenogeneic graft comparing to the CTG. In females, the ischemic period lasted for 7-12 days depending on the graft and no hyperemic response was observed. Females had more intense and prolonged wound fluid production. The LSCI method is suitable to capture the microcirculatory effect of the surgical intervention in human oral mucosa. The application of xenogeneic collagen matrices as a CTG substitute does not seem to restrain the recovery of graft bed circulation. Gender may have an effect on postoperative circulation and inflammation.

Adaptation of the cerebrocortical circulation to carotid artery occlusion involves blood flow redistribution between cortical regions and is independent of eNOS.

Cerebral circulation is secured by feed-forward and feed-back control pathways to maintain and eventually reestablish the optimal oxygen and nutrient supply of neurons in case of disturbances of the cardiovascular system. Using the high temporal and spatial resolution of laser-speckle imaging we aimed to analyze the pattern of cerebrocortical blood flow (CoBF) changes after unilateral (left) carotid artery occlusion (CAO) in anesthetized mice to evaluate the contribution of macrovascular (circle of Willis) vs. pial collateral vessels as well as that of endothelial nitric oxide synthase (eNOS) to the cerebrovascular adaptation to CAO. In wild-type mice CoBF reduction in the left temporal cortex started immediately after CAO, reaching its maximum (-26%) at 5-10 s. Thereafter, CoBF recovered close to the preocclusion level within 30 s indicating the activation of feed-back pathway(s). Interestingly, the frontoparietal cerebrocortical regions also showed CoBF reduction in the left (-17-19%) but not in the right hemisphere, although these brain areas receive their blood supply from the common azygos anterior cerebral artery in mice. In eNOS-deficient animals the acute CoBF reduction after CAO was unaltered, and the recovery was even accelerated compared with controls. These results indicate that 1) the Willis circle alone is not sufficient to provide an immediate compensation for the loss of one carotid artery, 2) pial collaterals attenuate the ischemia of the temporal cortex ipsilateral to CAO at the expense of the blood supply of the frontoparietal region, and 3) eNOS, surprisingly, does not play an important role in this CoBF redistribution.