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1. Evaluating cell viability, capillary perfusion, and collateral tortuosity in an ex vivo mouse intestine fluidics model

   https://doi.org/10.3389/fbioe.2022.1008481  

   Willi, Caroline E. ; Abdelazim, Hanaa ; Chappell, John C. ( December 2022 , Frontiers in Bioengineering and Biotechnology)

   Numerous disease conditions involve the sudden or progressive loss of blood flow. Perfusion restoration is vital for returning affected organs to full health. While a range of clinical interventions can successfully restore flow to downstream tissues, the microvascular responses after a loss-of-flow event can vary over time and may involve substantial microvessel instability. Increased insight into perfusion-mediated capillary stability and access-to-flow is therefore essential for advancing therapeutic reperfusion strategies and improving patient outcomes. To that end, we developed a tissue-based microvascular fluidics model to better understand (i) microvascular stability and access-to-flow over an acute time course post-ischemia, and (ii) collateral flow in vessels neighboring an occlusion site. We utilized murine intestinal tissue regions by catheterizing a feeder artery and introducing perfusate at physiologically comparable flow-rates. The cannulated vessel as well as a portion of the downstream vessels and associated intestinal tissue were cultured while constant perfusion conditions were maintained. An occlusion was introduced in a selected arterial segment, and changes in perfusion within areas receiving varying degrees of collateral flow were observed over time. To observe the microvascular response to perfusion changes, we incorporated (i) tissues harboring cell-reporter constructs, specifically Ng2-DsRed labeling of intestinal pericytes, and (ii) different types of fluorescent perfusates to quantify capillary access-to-flow at discrete time points. In our model, we found that perfusion tracers could enter capillaries within regions downstream of an occlusion upon the initial introduction of perfusion, but at 24 h tissue perfusion was severely decreased. However, live/dead cell discrimination revealed that the tissue overall did not experience significant cell death, including that of microvascular pericytes, even after 48 h. Our findings suggest that altered flow conditions may rapidly initiate cellular responses that reduce capillary access-to-flow, even in the absence of cellular deterioration or hypoxia. Overall, this ex vivo tissue-based microfluidics model may serve as a platform upon which a variety of follow-on studies may be conducted. It will thus enhance our understanding of microvessel stability and access-to-flow during an occlusive event and the role of collateral flow during normal and disrupted perfusion. 

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2. Metal Coordination Complexes as Therapeutic Agents for Ischemia-Reperfusion Injury

   https://doi.org/10.1021/jacs.3c01984  

   Bigham, Nicholas P. ; Wilson, Justin J. ( April 2023 , Journal of the American Chemical Society)

   Ischemia-reperfusion injury (IRI), which describes the cell damage and death that occurs after blood and oxygen are restored to ischemic or hypoxic tissue, is a significant factor within the mortality rates of heart disease and stroke patients. At the cellular level, the return of oxygen triggers an increase in reactive oxygen species (ROS) and mitochondrial calcium (mCa2+) overload, which both contribute to cell death. Despite the widespread occurrence of IRI in different pathological conditions, there are currently no clinically approved therapeutic agents for its management. In this Perspective, we will briefly discuss the current therapeutic options for IRI and then describe in great detail the potential role and arising applications of metal-containing coordination and organometallic complexes for treating this condition. This Perspective categorizes these metal compounds based on their mechanisms of action, which include their use as delivery agents for gasotransmitters, inhibitors of mCa2+ uptake, and catalysts for the decomposition of ROS. Lastly, the challenges and opportunities for inorganic chemistry approaches to manage IRI are discussed. 

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3. MiR-34a Interacts with Cytochrome c and Shapes Stroke Outcomes

   https://doi.org/10.1038/s41598-020-59997-y  

   Hu, Heng ; Hone, Emily A. ; Provencher, Edward A. P. ; Sprowls, Samuel A. ; Farooqi, Imran ; Corbin, Deborah R. ; Sarkar, Saumyendra N. ; Hollander, John M. ; Lockman, Paul R. ; Simpkins, James W. ; et al ( February 2020 , Scientific Reports)

   Blood-brain barrier (BBB) dysfunction occurs in cerebrovascular diseases and neurodegenerative disorders such as stroke. Opening of the BBB during a stroke has a negative impact on acute outcomes. We have recently demonstrated that miR-34a regulates the BBB by targeting cytochrome c (CYC)in vitro. To investigate the role of miR-34a in a stroke, we purified primary cerebrovascular endothelial cells (pCECs) from mouse brains following 1 h transient middle cerebral artery occlusion (tMCAO) and measured real-time PCR to detect miR-34a levels. We demonstrate that the miR-34a levels are elevated in pCECs from tMCAO mice at the time point of BBB opening following 1 h tMCAO and reperfusion. Interestingly, knockout of miR-34a significantly reduces BBB permeability, alleviates disruption of tight junctions, and improves stroke outcomes compared to wild-type (WT) controls. CYC is decreased in the ischemic hemispheres and pCECs from WT but not in miR-34a^−/−mice following stroke reperfusion. We further confirmed CYC is a target of miR-34a by a dural luciferase reporter gene assayin vitro. Our study provides the first description of miR-34a affecting stroke outcomes and may lead to discovery of new mechanisms and treatments for cerebrovascular and neurodegenerative diseases such as stroke.

    

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4. Differential effect of gold nanoparticles on cerebrovascular function and biomechanical properties

   https://doi.org/10.14814/phy2.15789  

   Hunt, Ryan D. ; Sedighi, Omid ; Clark, Wayne M. ; Doiron, Amber L. ; Cipolla, Marilyn J. ( August 2023 , Physiological Reports)

   Human stroke serum (HSS) has been shown to impair cerebrovascular function, likely by factors released into the circulation after ischemia. 20 nm gold nanoparticles (GNPs) have demonstrated anti‐inflammatory properties, with evidence that they decrease pathologic markers of ischemic severity. Whether GNPs affect cerebrovascular function, and potentially protect against the damaging effects of HSS on the cerebral circulation remains unclear. HSS obtained 24 h poststroke was perfused through the lumen of isolated and pressurized third‐order posterior cerebral arteries (PCAs) from male Wistar rats with and without GNPs (~2 × 10⁹GNP/ml), or GNPs in vehicle, in an arteriograph chamber (n = 8/group). All vessels were myogenically reactive ≥60 mmHg intravascular pressure; however, vessels containing GNPs had significantly less myogenic tone. GNPs increased vasoreactivity to small and intermediate conductance calcium activated potassium channel activation via NS309; however, reduced vasoconstriction to nitric oxide synthase inhibition. Hydraulic conductivity and transvascular filtration, were decreased by GNPs, suggesting a protective effect on the blood–brain barrier. The stress–strain curves of PCAs exposed to GNPs were shifted leftward, indicating increased vessel stiffness. This study provides the first evidence that GNPs affect the structure and function of the cerebrovasculature, which may be important for their development and use in biomedical applications.

    

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5. Peripherally misfolded proteins exacerbate ischemic stroke-induced neuroinflammation and brain injury

   https://doi.org/10.1186/s12974-021-02081-7  

   Liu, Yanying ; Subedi, Kalpana ; Baride, Aravind ; Romanova, Svetlana ; Callegari, Eduardo ; Huber, Christa C. ; Wang, Xuejun ; Wang, Hongmin ( December 2021 , Journal of Neuroinflammation)

   Abstract Background Protein aggregates can be found in peripheral organs, such as the heart, kidney, and pancreas, but little is known about the impact of peripherally misfolded proteins on neuroinflammation and brain functional recovery following ischemic stroke. Methods Here, we studied the ischemia/reperfusion (I/R) induced brain injury in mice with cardiomyocyte-restricted overexpression of a missense (R120G) mutant small heat shock protein, αB-crystallin (CryAB R120G ), by examining neuroinflammation and brain functional recovery following I/R in comparison to their non-transgenic (Ntg) littermates. To understand how peripherally misfolded proteins influence brain functionality, exosomes were isolated from CryAB R120G and Ntg mouse blood and were used to treat wild-type (WT) mice and primary cortical neuron-glia mix cultures. Additionally, isolated protein aggregates from the brain following I/R were isolated and subjected to mass-spectrometric analysis to assess whether the aggregates contained the mutant protein, CryAB R120G . To determine whether the CryAB R120G misfolding can self-propagate, a misfolded protein seeding assay was performed in cell cultures. Results Our results showed that CryAB R120G mice exhibited dramatically increased infarct volume, delayed brain functional recovery, and enhanced neuroinflammation and protein aggregation in the brain following I/R when compared to the Ntg mice. Intriguingly, mass-spectrometric analysis of the protein aggregates isolated from CryAB R120G mouse brains confirmed presence of the mutant CryAB R120G protein in the brain. Importantly, intravenous administration of WT mice with the exosomes isolated from CryAB R120G mouse blood exacerbated I/R-induced cerebral injury in WT mice. Moreover, incubation of the CryAB R120G mouse exosomes with primary neuronal cultures induced pronounced protein aggregation. Transduction of CryAB R120G aggregate seeds into cell cultures caused normal CryAB proteins to undergo dramatic aggregation and form large aggregates, suggesting self-propagation of CryAB R120G misfolding in cells. Conclusions These results suggest that peripherally misfolded proteins in the heart remotely enhance neuroinflammation and exacerbate brain injury following I/R likely through exosomes, which may represent an underappreciated mechanism underlying heart-brain crosstalk. 

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