Hypoxia-induced carbonic anhydrase mediated dorsal horn neuron activation and induction of neuropathic pain.

ABSTRACT: Neuropathic pain, such as that seen in diabetes mellitus, results in part from central sensitisation in the dorsal horn. However, the mechanisms responsible for such sensitisation remain unclear. There is evidence that disturbances in the integrity of the spinal vascular network can be causative factors in the development of neuropathic pain. Here we show that reduced blood flow and vascularity of the dorsal horn leads to the onset of neuropathic pain. Using rodent models (type 1 diabetes and an inducible endothelial-specific vascular endothelial growth factor receptor 2 knockout mouse) that result in degeneration of the endothelium in the dorsal horn, we show that spinal cord vasculopathy results in nociceptive behavioural hypersensitivity. This also results in increased hypoxia in dorsal horn neurons, depicted by increased expression of hypoxia markers such as hypoxia inducible factor 1α, glucose transporter 3, and carbonic anhydrase 7. Furthermore, inducing hypoxia through intrathecal delivery of dimethyloxalylglycine leads to the activation of dorsal horn neurons as well as mechanical and thermal hypersensitivity. This shows that hypoxic signalling induced by reduced vascularity results in increased hypersensitivity and pain. Inhibition of carbonic anhydrase activity, through intraperitoneal injection of acetazolamide, inhibited hypoxia-induced pain behaviours. This investigation demonstrates that induction of a hypoxic microenvironment in the dorsal horn, as occurs in diabetes, is an integral process by which neurons are activated to initiate neuropathic pain states. This leads to the conjecture that reversing hypoxia by improving spinal cord microvascular blood flow could reverse or prevent neuropathic pain.

18F-GP1 Positron Emission Tomography and Bioprosthetic Aortic Valve Thrombus.

BACKGROUND: Bioprosthetic valve thrombosis may have implications for valve function and durability. OBJECTIVES: Using a novel glycoprotein IIb/IIIa receptor radiotracer 18F-GP1, we investigated whether positron emission tomography (PET)-computed tomography (CT) could detect thrombus formation on bioprosthetic aortic valves. METHODS: Ex vivo experiments were performed on human platelets and explanted bioprosthetic aortic valves. In a prospective cross-sectional study, patients with either bioprosthetic or normal native aortic valves underwent echocardiography, CT angiography, and 18F-GP1 PET-CT. RESULTS: Flow cytometric analysis, histology, immunohistochemistry, and autoradiography demonstrated selective binding of 18F-GP1 to activated platelet glycoprotein IIb/IIIa receptors and thrombus adherent to prosthetic valves. In total, 75 participants were recruited: 53 with bioprosthetic valves (median time from implantation 37 months [IQR: 12-80 months]) and 22 with normal native aortic valves. Three participants had obstructive valve thrombosis, and a further 3 participants had asymptomatic hypoattenuated leaflet thickening on CT angiography. All bioprosthetic valves, but none of the native aortic valves, demonstrated focal 18F-GP1 uptake on the valve leaflets: median maximum target-to-background ratio 2.81 (IQR: 2.29-3.48) vs 1.43 (IQR: 1.28-1.53) (P < 0.001). Higher 18F-GP1 uptake was independently associated with duration of valve implantation and hypoattenuated leaflet thickening. All 3 participants with obstructive valve thrombosis were anticoagulated for 3 months, leading to resolution of their symptoms, improvement in mean valve gradients, and a reduction in 18F-GP1 uptake. CONCLUSIONS: Adherence of activated platelets is a common and sustained finding on bioprosthetic aortic valves. 18F-GP1 uptake is higher in the presence of thrombus, regresses with anticoagulation, and has potential use as an adjunctive clinical tool. (18F-GP1 PET-CT to Detect Bioprosthetic Aortic Valve Thrombosis; NCT04073875).

Aligned Poly-l-lactic Acid Nanofibers Induce Self-Assembly of Primary Cortical Neurons into 3D Cell Clusters.

Relative to two-dimensional (2D) culture, three-dimensional (3D) culture of primary neurons has yielded increasingly physiological responses from cells. Electrospun nanofiber scaffolds are frequently used as a 3D biomaterial support for primary neurons in neural tissue engineering, while hydrophobic surfaces typically induce aggregation of cells. Poly-l-lactic acid (PLLA) was electrospun as aligned PLLA nanofiber scaffolds to generate a structure with both qualities. Primary cortical neurons from E18 Sprague-Dawley rats cultured on aligned PLLA nanofibers generated 3D clusters of cells that extended highly aligned, fasciculated neurite bundles within 10 days. These clusters were viable for 28 days and responsive to AMPA and GABA. Relative to the 2D culture, the 3D cultures exhibited a more developed profile; mass spectrometry demonstrated an upregulation of proteins involved in cortical lamination, polarization, and axon fasciculation and a downregulation of immature neuronal markers. The use of artificial neural network inference suggests that the increased formation of synapses may drive the increase in development that is observed for the 3D cell clusters. This research suggests that aligned PLLA nanofibers may be highly useful for generating advanced 3D cell cultures for high-throughput systems.

Prostaglandin E2 Dilates Intracerebral Arterioles When Applied to Capillaries: Implications for Small Vessel Diseases.

Prostaglandin E2 (PGE2) has been widely proposed to mediate neurovascular coupling by dilating brain parenchymal arterioles through activation of prostanoid EP4 receptors. However, our previous report that direct application of PGE2 induces an EP1-mediated constriction strongly argues against its direct action on arterioles during neurovascular coupling, the mechanisms sustaining functional hyperemia. Recent advances have highlighted the role of capillaries in sensing neuronal activity and propagating vasodilatory signals to the upstream penetrating parenchymal arteriole. Here, we examined the effect of capillary stimulation with PGE2 on upstream arteriolar diameter using an ex vivo capillary-parenchymal arteriole preparation and in vivo cerebral blood flow measurements with two-photon laser-scanning microscopy. We found that PGE2 caused upstream arteriolar dilation when applied onto capillaries with an EC50 of 70 nM. The response was inhibited by EP1 receptor antagonist and was greatly reduced, but not abolished, by blocking the strong inward-rectifier K+ channel. We further observed a blunted dilatory response to capillary stimulation with PGE2 in a genetic mouse model of cerebral small vessel disease with impaired functional hyperemia. This evidence casts previous findings in a different light, indicating that capillaries are the locus of PGE2 action to induce upstream arteriolar dilation in the control of brain blood flow, thereby providing a paradigm-shifting view that nonetheless remains coherent with the broad contours of a substantial body of existing literature.

Coronary 18F-Fluoride Uptake and Progression of Coronary Artery Calcification.

Background Positron emission tomography (PET) using 18F-sodium fluoride (18F-fluoride) to detect microcalcification may provide insight into disease activity in coronary atherosclerosis. This study aimed to investigate the relationship between 18F-fluoride uptake and progression of coronary calcification in patients with clinically stable coronary artery disease. Methods Patients with established multivessel coronary atherosclerosis underwent 18F-fluoride PET-computed tomography angiography and computed tomography calcium scoring, with repeat computed tomography angiography and calcium scoring at one year. Coronary PET uptake was analyzed qualitatively and semiquantitatively in diseased vessels by measuring maximum tissue-to-background ratio. Coronary calcification was quantified by measuring calcium score, mass, and volume. Results In a total of 183 participants (median age 66 years, 80% male), 116 (63%) patients had increased 18F-fluoride uptake in at least one vessel. Individuals with increased 18F-fluoride uptake demonstrated more rapid progression of calcification compared with those without uptake (change in calcium score, 97 [39-166] versus 35 [7-93] AU; P<0.0001). Indeed, the calcium score only increased in coronary segments with 18F-fluoride uptake (from 95 [30-209] to 148 [61-289] AU; P<0.001) and remained unchanged in segments without 18F-fluoride uptake (from 46 [16-113] to 49 [20-115] AU; P=0.329). Baseline coronary 18F-fluoride maximum tissue-to-background ratio correlated with 1-year change in calcium score, calcium volume, and calcium mass (Spearman ρ=0.37, 0.38, and 0.46, respectively; P<0.0001 for all). At the segmental level, baseline 18F-fluoride activity was an independent predictor of calcium score at 12 months (P<0.001). However, at the patient level, this was not independent of age, sex, and baseline calcium score (P=0.50). Conclusions Coronary 18F-fluoride uptake identifies both patients and individual coronary segments with more rapid progression of coronary calcification, providing important insights into disease activity within the coronary circulation. At the individual patient level, total calcium score remains an important marker of disease burden and progression. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02110303.

The Ion Channel and GPCR Toolkit of Brain Capillary Pericytes.

Brain pericytes reside on the abluminal surface of capillaries, and their processes cover ~90% of the length of the capillary bed. These cells were first described almost 150 years ago (Eberth, 1871; Rouget, 1873) and have been the subject of intense experimental scrutiny in recent years, but their physiological roles remain uncertain and little is known of the complement of signaling elements that they employ to carry out their functions. In this review, we synthesize functional data with single-cell RNAseq screens to explore the ion channel and G protein-coupled receptor (GPCR) toolkit of mesh and thin-strand pericytes of the brain, with the aim of providing a framework for deeper explorations of the molecular mechanisms that govern pericyte physiology. We argue that their complement of channels and receptors ideally positions capillary pericytes to play a central role in adapting blood flow to meet the challenge of satisfying neuronal energy requirements from deep within the capillary bed, by enabling dynamic regulation of their membrane potential to influence the electrical output of the cell. In particular, we outline how genetic and functional evidence suggest an important role for Gs-coupled GPCRs and ATP-sensitive potassium (KATP) channels in this context. We put forth a predictive model for long-range hyperpolarizing electrical signaling from pericytes to upstream arterioles, and detail the TRP and Ca2+ channels and Gq, Gi/o, and G12/13 signaling processes that counterbalance this. We underscore critical questions that need to be addressed to further advance our understanding of the signaling topology of capillary pericytes, and how this contributes to their physiological roles and their dysfunction in disease.

Substantial Metabolic Activity of Human Brown Adipose Tissue during Warm Conditions and Cold-Induced Lipolysis of Local Triglycerides.

Current understanding of in vivo human brown adipose tissue (BAT) physiology is limited by a reliance on positron emission tomography (PET)/computed tomography (CT) scanning, which has measured exogenous glucose and fatty acid uptake but not quantified endogenous substrate utilization by BAT. Six lean, healthy men underwent 18fluorodeoxyglucose-PET/CT scanning to localize BAT so microdialysis catheters could be inserted in supraclavicular BAT under CT guidance and in abdominal subcutaneous white adipose tissue (WAT). Arterial and dialysate samples were collected during warm (∼25°C) and cold exposure (∼17°C), and blood flow was measured by 133xenon washout. During warm conditions, there was increased glucose uptake and lactate release and decreased glycerol release by BAT compared with WAT. Cold exposure increased blood flow, glycerol release, and glucose and glutamate uptake only by BAT. This novel use of microdialysis reveals that human BAT is metabolically active during warm conditions. BAT activation substantially increases local lipolysis but also utilization of other substrates such as glutamate.

Quality control within the multicentre perfusion CT study of primary colorectal cancer (PROSPeCT): results of an iodine density phantom study.

OBJECTIVES: To assess the cross-centre consistency of iodine enhancement, contrast-to-noise ratio and radiation dose in a multicentre perfusion CT trial of colorectal cancer. MATERIALS AND METHODS: A cylindrical water phantom containing different iodine inserts was examined on seven CT models in 13 hospitals. The relationship between CT number (Hounsfield units, HU) and iodine concentration (milligrams per millilitre) was established and contrast-to-noise ratios (CNRs) calculated. Radiation doses (CTDIvol, DLP) were compared across all sites. RESULTS: There was a linear relationship between CT number and iodine density. Iodine enhancement varied by a factor of at most 1.10, and image noise by at most 1.5 across the study sites. At an iodine concentration of 1 mg ml(-1) and 100 kV, CNRs ranged from 3.6 to 4.8 in the 220-mm phantom and from 1.4 to 1.9 in the 300-mm phantom. Doses varied by a factor of at most 2.4, but remained within study dose constraints. Iterative reconstruction algorithms did not alter iodine enhancement but resulted in reduced image noise by a factor of at most 2.2, allowing a potential dose decrease of at most 80% compared to filtered back projection (FBP). CONCLUSIONS: Quality control of CT performance across centres indicates that CNR values remain relatively consistent across all sites, giving acceptable image quality within the agreed dose constraints. KEY POINTS: Quality control is essential in a multicentre setting to enable CT quantification. CNRs in a body-sized phantom had the recommended value of at least 1.5. CTDIs and DLPs varied by factors of 1.8 and 2.4 respectively.

A systematic review of the utility of 1.5 versus 3 Tesla magnetic resonance brain imaging in clinical practice and research.

OBJECTIVE: MRI at 3 T is said to be more accurate than 1.5 T MR, but costs and other practical differences mean that it is unclear which to use. METHODS: We systematically reviewed studies comparing diagnostic accuracy at 3 T with 1.5 T. We searched MEDLINE, EMBASE and other sources from 1 January 2000 to 22 October 2010 for studies comparing diagnostic accuracy at 1.5 and 3 T in human neuroimaging. We extracted data on methodology, quality criteria, technical factors, subjects, signal-to-noise, diagnostic accuracy and errors according to QUADAS and STARD criteria. RESULTS: Amongst 150 studies (4,500 subjects), most were tiny, compared old 1.5 T with new 3 T technology, and only 22 (15 %) described diagnostic accuracy. The 3 T images were often described as "crisper", but we found little evidence of improved diagnosis. Improvements were limited to research applications [functional MRI (fMRI), spectroscopy, automated lesion detection]. Theoretical doubling of the signal-to-noise ratio was not confirmed, mostly being 25 %. Artefacts were worse and acquisitions took slightly longer at 3 T. CONCLUSION: Objective evidence to guide MRI purchasing decisions and routine diagnostic use is lacking. Rigorous evaluation accuracy and practicalities of diagnostic imaging technologies should be the routine, as for pharmacological interventions, to improve effectiveness of healthcare. KEY POINTS : • Higher field strength MRI may improve image quality and diagnostic accuracy. • There are few direct comparisons of 1.5 and 3 T MRI. • Theoretical doubling of the signal-to-noise ratio in practice was only 25 %. • Objective evidence of improved routine clinical diagnosis is lacking. • Other aspects of technology improved images more than field strength.