Cortical Microvascular Perfusion Research Articles

Neuroprotective effect of the RNS60 in a mouse model of transient focal cerebral ischemia.

Stroke is a major cause of death, disability, and public health problems. Its intervention is limited to early treatment with thrombolytics and/or endovascular clot removal with mechanical thrombectomy without any available subacute or chronic neuroprotective treatments. RNS60 has reduced neuroinflammation and increased neuronal survival in several animal models of neurodegeneration and trauma. The aim here was to evaluate whether RNS60 protects the brain and cognitive function in a mouse stroke model. Male C57BL/6J mice were subjected to sham or ischemic stroke surgery using 60-minute transient middle cerebral artery occlusion (tMCAo). In each group, mice received blinded daily administrations of RNS60 or control fluids (PNS60 or normal saline [NS]), beginning 2 hours after surgery over 13 days. Multiple neurobehavioral tests were conducted (Neurological Severity Score [mNSS], Novel Object Recognition [NOR], Active Place Avoidance [APA], and the Conflict Variant of APA [APAc]). On day 14, cortical microvascular perfusion (MVP) was measured, then brains were removed and infarct volume, immunofluorescence of amyloid beta (Aβ), neuronal density, microglial activation, and white matter damage/myelination were measured. SPSS was used for analysis (e.g., ANOVA for parametric data; Kruskal Wallis for non-parametric data; with post-hoc analysis). Thirteen days of treatment with RNS60 reduced brain infarction, amyloid pathology, neuronal death, microglial activation, white matter damage, and increased MVP. RNS60 reduced brain pathology and resulted in behavioral improvements in stroke compared to sham surgery mice (increased memory-learning in NOR and APA, improved cognitive flexibility in APAc). RNS60-treated mice exhibit significant protection of brain tissue and improved neurobehavioral functioning after tMCAo-stroke. Additional work is required to determine mechanisms, time-window of dosing, and multiple dosing volumes durations to support clinical stroke research.

Prediction of renal recovery following sepsis-associated acute kidney injury requiring renal replacement therapy using contrast-enhanced ultrasonography.

Microcirculatory dysfunction plays a critical role in sepsis-associated acute kidney injury (S-AKI) development; however, its impact on renal recovery remains uncertain. We investigated the association between cortical microcirculatory function assessed using contrast-enhanced ultrasonography (CEUS) and renal recovery after S-AKI needing renal replacement therapy (RRT). This retrospective study included 23 patients who underwent CEUS among those who underwent acute RRT for S-AKI. In addition, we acquired data from 17 healthy individuals and 18 patients with chronic kidney disease. Renal recovery was defined as sustained independence from RRT for at least 14 days. Of the CEUS-derived parameters, rise time, time to peak, and fall time were longer in patients with S-AKI than in healthy individuals (p = 0.045, 0.01, and 0.096, respectively). Fourteen patients (60.9%) with S-AKI receiving RRT experienced renal recovery; and these patients had higher values of peak enhancement, wash-in area under the curve (AUC), wash-in perfusion index, and washout AUC than those without recovery (p = 0.03, 0.01, 0.03, and 0.046, respectively). We evaluated the receiver operating characteristic curve and found that the peak enhancement, wash-in AUC, wash-in perfusion index, and wash-out AUC of CEUS derivatives estimated the probability of renal recovery after S-AKI requiring RRT (p = 0.03, 0.01, 0.03, and 0.04, respectively). CEUS-assessed cortical microvascular perfusion may predict renal recovery following S-AKI that requires RRT. Further studies are essential to validate the clinical utility of microcirculatory parameters obtained from CEUS to estimate renal outcomes in various etiologies and severities of kidney disease.

Abstract TP219: Rns60 Brain Protection In Mouse Ischemic Stroke Mitigates Plasma Tau And Cortical Microvascular Perfusion

Introduction: RNS60 is 0.9% saline solution hypothesized to contain oxygen nanobubbles. Recently, we showed that 13 days of RNS60 treatment after transient middle cerebral artery occlusion (tMCAo) preserves recovery of memory and reduces brain infarction, neuronal cell death, microglial activation, amyloid pathology, and white matter damage. Previously, we also demonstrated the relationship between white matter demyelination and circulating tau in tMCAo. Here we report that RNS60 decreases plasma tau and increases cortical microvascular perfusion (MVP). Methods: Male C57BL/6J mice, 3-4 months old, were randomly divided into sham surgery or unilateral 60-minute tMCAo. Each group was subdivided into three treatment arms, receiving daily intraperitoneal administration of 0.2 mL of RNS60, pressurized normal saline (PNS60), or normal saline (NS) starting 2 hours after surgery for 13 days. Treatment assignments were blinded throughout the study. To assess circulating tau, plasma was collected before surgery and on day 14 post-surgery (after MVP measurements). Animals were then euthanized to assess infarct volume using TTC staining. Results: RNS60 treatment significantly (p<0.05) reduced brain infarction by 90% compared to controls, reduced plasma tau by 74% of controls, and significantly (p<0.05) increased MVP in motor cortex by 38% and by 39% in the parietal cortex. Conclusion: RNS60-treated mice exhibit significant brain protection after ischemic stroke and display decreased plasma tau levels and a significant increase in microvascular perfusion. Ongoing studies are investigating whether increased MVP is due to activation of angiogenesis. These data further support the evaluation of RNS60 in clinical stroke trials.

Association of Contrast-Enhanced Ultrasound-Derived Kidney Cortical Microvascular Perfusion with Kidney Function.

Individuals with chronic kidney disease (CKD) have decreased kidney cortical microvascular perfusion, which may lead to worsening kidney function over time, but methods to quantify kidney cortical microvascular perfusion are not feasible to incorporate into clinical practice. Contrast-enhanced ultrasound (CEUS) may quantify kidney cortical microvascular perfusion, which requires further investigation in individuals across the spectrum of kidney function. We performed CEUS on a native kidney of 83 individuals across the spectrum of kidney function and calculated quantitative CEUS-derived kidney cortical microvascular perfusion biomarkers. Participants had a continuous infusion of the microbubble contrast agent (Definity) with a flash-replenishment sequence during their CEUS scan. Lower values of the microbubble velocity (β) and perfusion index (β×A) may represent lower kidney cortical microvascular perfusion. Multivariable linear regression models tested the associations of the microbubble velocity (β) and perfusion index (β×A) with estimated glomerular filtration rate (eGFR). Thirty-eight individuals with CKD (mean age±SD 65.2±12.6 years, median [IQR] eGFR 31.5 [18.9-41.5] ml/min per 1.73 m2), 37 individuals with end stage kidney disease (ESKD; age 54.8±12.3 years), and eight healthy volunteers (age 44.1±15.0 years, eGFR 117 [106-120] ml/min per 1.73 m2) underwent CEUS without side effects. Individuals with ESKD had the lowest microbubble velocity (β) and perfusion index (β×A) compared with individuals with CKD and healthy volunteers. The microbubble velocity (β) and perfusion index (β×A) had moderate positive correlations with eGFR (β: rs=0.44, P<0.001; β×A: rs=0.50, P<0.001). After multivariable adjustment, microbubble velocity (β) and perfusion index (β×A) remained significantly associated with eGFR (change in natural log transformed eGFR per 1 unit increase in natural log transformed biomarker: β, 0.38 [95%, CI 0.17 to 0.59]; β×A, 0.79 [95% CI, 0.45 to 1.13]). CEUS-derived kidney cortical microvascular perfusion biomarkers are associated with eGFR. Future studies are needed to determine if CEUS-derived kidney cortical microvascular perfusion biomarkers have prognostic value.

Increases in microvascular perfusion and tissue oxygenation via pulsed electromagnetic fields in the healthy rat brain.

High-frequency pulsed electromagnetic field stimulation is an emerging noninvasive therapy being used clinically to facilitate bone and cutaneous wound healing. Although the mechanisms of action of pulsed electromagnetic fields (PEMF) are unknown, some studies suggest that its effects are mediated by increased nitric oxide (NO), a well-known vasodilator. The authors hypothesized that in the brain, PEMF increase NO, which induces vasodilation, enhances microvascular perfusion and tissue oxygenation, and may be a useful adjunct therapy in stroke and traumatic brain injury. To test this hypothesis, they studied the effect of PEMF on a healthy rat brain with and without NO synthase (NOS) inhibition. In vivo two-photon laser scanning microscopy (2PLSM) was used on the parietal cortex of rat brains to measure microvascular tone and red blood cell (RBC) flow velocity in microvessels with diameters ranging from 3 to 50 μm, which includes capillaries, arterioles, and venules. Tissue oxygenation (reduced nicotinamide adenine dinucleotide [NADH] fluorescence) was also measured before and for 3 hours after PEMF treatment using the FDA-cleared SofPulse device (Ivivi Health Sciences, LLC). To test NO involvement, the NOS inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME) was intravenously injected (10 mg/kg). In a time control group, PEMF were not used. Doppler flux (0.8-mm probe diameter), brain and rectal temperatures, arterial blood pressure, blood gases, hematocrit, and electrolytes were monitored. Pulsed electromagnetic field stimulation significantly dilated cerebral arterioles from a baseline average diameter of 26.4 ± 0.84 μm to 29.1 ± 0.91 μm (11 rats, p < 0.01). Increased blood volume flow through dilated arterioles enhanced capillary flow with an average increase in RBC flow velocity by 5.5% ± 1.3% (p < 0.01). Enhanced microvascular flow increased tissue oxygenation as reflected by a decrease in NADH autofluorescence to 94.7% ± 1.6% of baseline (p < 0.05). Nitric oxide synthase inhibition by L-NAME prevented PEMF-induced changes in arteriolar diameter, microvascular perfusion, and tissue oxygenation (7 rats). No changes in measured parameters were observed throughout the study in the untreated time controls (5 rats). This is the first demonstration of the acute effects of PEMF on cerebral cortical microvascular perfusion and metabolism. Thirty minutes of PEMF treatment induced cerebral arteriolar dilation leading to an increase in microvascular blood flow and tissue oxygenation that persisted for at least 3 hours. The effects of PEMF were mediated by NO, as we have shown in NOS inhibition experiments. These results suggest that PEMF may be an effective treatment for patients after traumatic or ischemic brain injury. Studies on the effect of PEMF on the injured brain are in progress.

Real-time assessment of renal cortical microvascular perfusion heterogeneities using near-infrared laser speckle imaging

Laser speckle imaging (LSI) is able to provide full-field perfusion maps of the renal cortex and allows quantification of the average LSI perfusion within an arbitrarily set region of interest and the recovery of LSI perfusion histograms within this region. The aim of the present study was to evaluate the use of LSI for mapping renal cortical microvascular perfusion and to demonstrate the capability of LSI to assess renal perfusion heterogeneities. The main findings were that: 1) full-field LSI measurements of renal microvascular perfusion were highly correlated to single-point LDV measurements; 2) LSI is able to detect differences in reperfusion dynamics following different durations of ischemia; and 3) renal microvascular perfusion heterogeneities can be quantitatively assessed by recovering LSI perfusion histograms.

Administration of transforming growth factor-alpha reduces infarct volume after transient focal cerebral ischemia in the rat.

Growth factors promote cell growth and survival and protect the brain from developing injury after ischemia. In this article, the authors examined whether transforming growth factor-alpha (TGF-alpha) was protective in transient focal ischemia and whether alteration of cerebral circulation was involved. Rats received intraventricular TGF-alpha (50 ng, either split into 2 doses given 30 minutes before and 30 minutes after middle cerebral artery occlusion (MCAO), or 1 dose given 30 minutes after MCAO) or vehicle. Rats were subjected to 1-hour intraluminal MCAO and cerebral blood flow was recorded continuously by laser-Doppler flowmetry. Infarct volume was measured 1 and 4 days later. The effects of TGF-alpha on arterial tone were assessed in isolated rabbit basilar and common carotid arteries. Transforming growth factor-alpha before and after ischemia reduced infarct volume by 70% at 1 day and 50% at 4 days. Transforming growth factor-alpha given only after ischemia also did reduce infarct volume by 70% at 1 day and 80% at 4 days. The protective effect was more marked in cortex than in striatum. Transforming growth factor-alpha did not change cortical microvascular perfusion and did not modify arterial passive tone nor agonist-induced active tone. It can be concluded that TGF-alpha reduces infarct volume, even when the factor is exclusively administered at reperfusion, and that this effect is not mediated by changes in microvascular perfusion or cerebral arteries. It is therefore suggested that TGF-alpha has a protective effect against neuronal cell death after transient focal ischemia.

Mouse Strain Differences in Susceptibility to Cerebral Ischemia are Related to Cerebral Vascular Anatomy

The consequences of cerebral ischemia were studied in three different strains (BDF, CFW, and BALB/C) of mice. The different strains exhibited significant differences in susceptibility to 24-h focal ischemia. Following middle cerebral artery occlusion (MCAO), infarct volumes (mm3) were 5 +/- 3 in BDF, 15 +/- 5 in CFW, and 23 +/- 3 in BALB/C mice (p < 0.05). MCAO plus ipsilateral common carotid artery occlusion (CCAO) resulted in infarct volumes of 15 +/- 9 in BDF, 38 +/- 10 in CFW, and 72 +/- 12 in BALB/C mice (p < 0.05). In addition, MCAO plus CCAO produced death by 24 h in 42% of CFW and 67% of BALB/C mice, but not in any BDF mice (p < 0.05). CCAO alone produced multifocal hemispheric infarctions in 36% of BALB/C mice but not in the other two strains. Brains of all mouse strains subjected to sham surgery were free of any ischemic injury. Arterial blood pressures, blood gases, and blood cell profiles were relatively similar for the three mouse strains. However, carbon black studies of the cerebrovascular anatomy revealed an incomplete circle of Willis (i.e., a significant decrease in the frequency of patent posterior communicating arteries) for BALB/C compared with BDF mice (p < 0.05), with CFW mice being intermediary. Based on these anatomical data, BALB/C mice also were evaluated following transient global brain ischemia produced by bilateral CCAO. BALB/C mice exhibited a > 85% reduction in cortical microvascular perfusion and EEG power within 1 min of bilateral CCAO. Also, hippocampal neuronal CA1 damage and mortality over 7 days were related to the duration of global brain ischemia (p < 0.05). These data demonstrate a significant difference between mouse strains in their sensitivity to cerebral ischemia that appears to be related, at least in part, to the functional vascular anatomy at the level of the posterior communicating arteries. In particular, we point out the potential usefulness of BALB/C mice as a sensitive and reproducible model of focal and global ischemia.

Heptyl-physostigmine enhances basal forebrain control of cortical cerebral blood flow.

This study sought to determine the effect of heptylphysostigmine (H-PHY), a reversible cholinesterase (ChE) inhibitor with greater lipophilicity and longer duration of action than physostigmine, on resting and basal forebrain (BF)-elicited increases in cortical cerebral blood flow (CBF). Laser-doppler flowmetry (LDF) was used to monitor changes in frontal cortical microvascular perfusion in urethane anesthetized rats. Responses were measured before, early after, and 1 hr following H-PHY, 3 mg/kg, i.m. Electrical stimulation (100 microA) of the BF elicited up to 220% increases in CBF at 50 Hz, an effect that was graded with frequency. At 15 min following H-PHY (3 mg/kg) resting cortical CBF was unchanged, whereas BF-elicited increases were potentiated 47% at 50 Hz. At 1 hour, resting cortical CBF remained unchanged, and the BF-elicited responses were remarkably potentiated by 354% at 10 Hz and 67% at 50 Hz. Acetylcholinesterase (AChE) activity measured in the tissue directly beneath the LDF probe was decreased by 84% at a time when these CBF responses were enhanced. These data suggest that H-PHY substantially enhances the regulation of cortical CBF by the BF, an effect that may be linked to inhibition of cortical AChE activity. This enhancement of cortical CBF may contribute to the efficacy of H-PHY as a treatment for Alzheimer's disease.

Cortical cerebral blood flow governed by the basal forebrain: Age-related Impairments

This study sought to compare resting and evoked increases in cortical microvascular perfusion elicited by electrical microstimulation of the basal forebrain (BF) in young (4–6 months) and aged (22–26 months) Sprague-Dawley rats. Regional cerebral blood flow (rCBF) was measured in chloralose-anesthetized rats for twelve bilateral regions using 14C-iodoantipyrine with regional brain dissection, while second-to-second changes in tissue perfusion were concurrently assessed using laser-doppler flowmetry (LDF). In young animals, BF stimulation elicited significant ipsilateral increases in CBF in parietal (+123%) and frontal (+107%) cortices, caudate nucleus (+63%) and thalamus (+59%) ( p<0.05). The BF-elicited increases were preserved in frontal cortex and thalamus, but not in parietal cortex or caudate nucleus of aged animals. No frequency- or current-specific attenuations were observed in the spared frontal cortex of aged animals. However, there was a significant (+70%) age-related increase in the latency to reach maximal blood flow increases ( p<0.05), without any change in the total time of increased blood flow. These findings support the hypothesis that cortical CBF is in part governed by BF neurons, and suggest that regionally selective, age-related impairments of cortical coupling of neuronal to dynamic vascular responses exist. It remains to be determined whether the mechanism of this impairment relates to an age-related impairment in coupling of blood flow and metabolism.

Abluminal effects of endothelin in cerebral microvasculature assessed by laser-Doppler flowmetry

Laser-Doppler flowmetry was used to assess the intraparenchymal effects of endothelin 1 (ET-1) on cortical microvascular perfusion (CP). The initial part of this study examined effects of the intraparenchymal microinjection technique on local cortical microvascular responsivity. In anesthetized rats, the microinjection of vehicle (saline) beneath the cortical surface did not alter CO2 responsivity or autoregulation of the cortical microvasculature. In addition, predictable monophasic changes in local CP were elicited by the intraparenchymal microinjection of known vasodilators and vasoconstrictors, i.e., nitroprusside and prostaglandin F2 alpha, respectively. These experiments demonstrate normal responsivity of the cortical microvasculature after intraparenchymal microinjection. In the second part of this study, intraparenchymal microinjections (100 nl) of ET-1 (1-1,000 fmol) were evaluated. Microinjections of less than 10 fmol of ET-1 did not alter CP. However, doses between 10 and 1,000 fmol of ET-1 elicited monophasic dose-related reductions in CP. At 1,000 fmol, the highest dose studied, ET-1 produced a complex microvascular response consisting of an initial profound reduction in CP followed by alternating cycles of increased perfusion ultimately lapsing back to a prolonged period of hypoperfusion. No significant changes in blood pressure were observed after ET-1 administration, and no significant changes in any of the hemodynamic variables were observed after vehicle microinjection. These results suggest that abluminal microvascular actions of ET-1 mediate cerebral vasoconstriction and hypoperfusion.

Effect of endothelin on cortical microvascular perfusion in rats.

We used laser-Doppler flowmetry to study the effects of endothelin-1 on local cortical microvascular perfusion and resistance in 29 pentobarbital-anesthetized rats. Intravenous administration of 10-300 pmol endothelin-1 reduced arterial blood pressure and microvascular resistance and increased microvascular perfusion. However, intracarotid administration of low doses of endothelin-1 increased microvascular perfusion and reduced microvascular resistance and arterial blood pressure, whereas high doses (greater than or equal to 300 pmol) reduced microvascular perfusion and increased microvascular resistance and arterial blood pressure. Only the high dose/low flow response was associated with attenuation of the electrocorticogram. The low dose/high flow and high dose/low flow responses to endothelin-1 were not altered by blockade of muscarinic and adrenergic receptors. In addition, systemic metabolic changes (arterial pH, PaCO2, PaO2, and plasma glucose concentration) did not account for the cerebrovascular effects of endothelin-1. Platelet hyperaggregability also did not appear to be a causative factor in the high dose/low flow response to endothelin-1. In fact, ex vivo rat platelet aggregation was inhibited by intracarotid administration of 300 pmol endothelin-1. In conclusion, the cerebral vasculature exhibits extreme sensitivity to the vasodilator properties of endothelin-1 at low doses. The ischemic vasoconstrictor effects observed at high doses implicate endothelin-1 as an important mediator of cerebral vasospasm and/or postischemic hypoperfusion.