Oophorectomy Reduces Estradiol Levels and Long-Term Spontaneous Neurovascular Recovery in a Female Rat Model of Focal Ischemic Stroke

Although epidemiological evidence suggests significant sex and gender-based differences in stroke risk and recovery, females have been widely under-represented in preclinical stroke research. The neurovascular sequelae of brain ischemia in females, in particular, are largely uncertain. We set out to address this gap by a multimodal in vivo study of neurovascular recovery from endothelin-1 model of cortical focal-stroke in sham vs. ovariectomized female rats. Three weeks post ischemic insult, sham operated females recapitulated the phenotype previously reported in male rats in this model, of normalized resting perfusion but sustained peri-lesional cerebrovascular hyperreactivity. In contrast, ovariectomized (Ovx) females showed reduced peri-lesional resting blood flow, and elevated cerebrovascular responsivity to hypercapnia in the peri-lesional and contra-lateral cortices. Electrophysiological recordings showed an attenuation of theta to low-gamma phase-amplitude coupling in the peri-lesional tissue of Ovx animals, despite relative preservation of neuronal power. Further, this chronic stage neuronal network dysfunction was inversely correlated with serum estradiol concentration. Our pioneering data demonstrate dramatic differences in spontaneous recovery in the neurovascular unit between Ovx and Sham females in the chronic stage of stroke, underscoring the importance of considering hormonal-dependent aspects of the ischemic sequelae in the development of novel therapeutic approaches and patient recruitment in clinical trials

Comparative characterization of Shiga toxin type 2 and subtilase cytotoxin effects on human renal epithelial and endothelial cells grown in monolayer and bilayer conditions

Published: June 23, 2016Postdiarrheal hemolytic uremic syndrome (HUS) affects children under 5 years old and is responsible for the development of acute and chronic renal failure, particularly in Argentina. This pathology is a complication of Shiga toxin (Stx)-producing Escherichia coli infection and renal damage is attributed to Stx types 1 and 2 (Stx1, Stx2) produced by Escherichia coli O157:H7 and many other STEC serotypes. It has been reported the production of Subtilase cytotoxin (SubAB) by non-O157 STEC isolated from cases of childhood diarrhea. Therefore, it is proposed that SubAB may contribute to HUS pathogenesis. The human kidney is the most affected organ because very Stx-sensitive cells express high amounts of biologically active receptor. In this study, we investigated the effects of Stx2 and SubAB on primary cultures of human glomerular endothelial cells (HGEC) and on a human tubular epithelial cell line (HK-2) in monoculture and coculture conditions. We have established the coculture as a human renal proximal tubule model to study water absorption and cytotoxicity in the presence of Stx2 and SubAB. We obtained and characterized cocultures of HGEC and HK-2. Under basal conditions, HGEC monolayers exhibited the lowest electrical resistance (TEER) and the highest water permeability, while the HGEC/HK-2 bilayers showed the highest TEER and the lowest water permeability. In addition, at times as short as 20-30 minutes, Stx2 and SubAB caused the inhibition of water absorption across HK-2 and HGEC monolayers and this effect was not related to a decrease in cell viability. However, toxins did not have inhibitory effects on water movement across HGEC/HK-2 bilayers. After 72 h, Stx2 inhibited the cell viability of HGEC and HK-2 monolayers, but these effects were attenuated in HGEC/HK-2 bilayers. On the other hand, SubAB cytotoxicity shows a tendency to be attenuated by the bilayers. Our data provide evidence about the different effects of these toxins on the bilayers respect to the monolayers. This in vitro model of communication between human renal microvascular endothelial cells and human proximal tubular epithelial cells is a representative model of the human proximal tubule to study the effects of Stx2 and SubAB related to the development of HUS.Romina S. Álvarez, Flavia Sacerdoti, Carolina Jancic, Adrienne W. Paton, James C. Paton, Cristina Ibarra, María M. Amara

Effect of vagus nerve stimulation on serotonergic and noradrenergic transmission

ABSTRACT Vagus nerve stimulation (VNS) is an antiepileptic treatment, which has recently shown promise as an antidepressant. Yet, its antidepressant mechanisms of action are unknown. Serotonergic [5-hydroxytryptamine (5-HT, serotonin)] and noradrenergic [norepinephrine (NE)] systems are involved in the pathophysiology of depression and in the mechanisms of action of antidepressants. The present study analyzes 5-HT and NE neuronal firing rates in their brainstem nuclei: the dorsal raphe nucleus (DRN) and locus coeruleus (LC), respectively. The basal firing rates in the DRN and LC were significantly increased after long-term treatments with VNS. After short-term VNS treatments, firing rates were significantly higher for LC (at 1 h and 3 days). As changes in their firing rate may have been due to altered autoreceptor sensitivities, the responses of autoreceptors to the acute administration of their respective agonists were assessed. However, no significant difference was seen in the DRN. No significant differences in dose response curves for 5-HT 1A somatodendritic and ␣ 2 -adrenergic autoreceptors were noticed between long-term VNS and controls. VNS appears to have a novel mechanism of antidepressant action, enabling its effectiveness in treatment-resistant depression. LC firing rates significantly increase earlier than the DRN basal firing. As the LC has an excitatory influence on DRN, it is possible that the increased DRN firing rate is secondary to an initial increased LC firing rate from VNS. The vagus nerve (cranial nerve 10) is generally thought of as a group of efferent parasympathetic fibers regulating autonomic functions. However, this nerve consists of 80% afferent fibers In addition to decreasing seizure frequency, an improvement in mood was witnessed in patients with vagus nerve stimulation (VNS), even in those with little or no change in seizure frequenc

Quantitative estimates of stimulation-induced perfusion response using two-photon fluorescence microscopy of cortical microvascular networks

Functional hyperemia, or the increase in focal perfusion elicited by neuronal activation, is one of the primary functions of the neurovascular unit and a hallmark of healthy brain functioning. While much is known about the hemodynamics on the millimeter to tenths of millimeter-scale accessible by MRI, there is a paucity of quantitative data on the micrometer-scale changes in perfusion in response to functional stimulation. We present a novel methodology for quantification of perfusion and intravascular flow across the 3D microvascular network in the rat somatosensory cortex using two-photon fluorescence microscopy (2PFM). For approximately 96% of responding microvessels in the forelimb representation of the primary somatosensory cortex, brief (~2 s) forepaw stimulation resulted in an increase of perfusion 20 ± 4% (mean ± sem). The perfusion levels associated with the remaining 4% of the responding microvessels decreased 10 ± 9% upon stimulation. Vessels irrigating regions of lower vascular density were found to exhibit higher flow (p b 0.02), supporting the notion that local vascular morphology and hemodynamics reflect the metabolic needs of the surrounding parenchyma. High dispersion (~77%) in perfusion levels suggests high spatial variation in tissue susceptibility to hypoxia. The current methodology enables quantification of absolute perfusion associated with individual vessels of the cortical microvascular bed and its changes in response to functional stimulation and thereby provides an important tool for studying the cellular mechanisms of functional hyperemia, the spatial specificity of perfusion response to functional stimulation, and, broadly, the micrometer-scale relationship between vascular morphology and function in health and disease

Altered cerebral blood flow and cerebrovascular function after voluntary exercise in adult mice

The beneficial effects of physical exercise on brain health are well documented, yet how exercise modulates cerebrovascular function is not well understood. This study used continuous arterial spin labeling magnetic resonance imaging with a hypercapnic challenge to examine changes in cerebral blood flow and vascular function after voluntary exercise in healthy, adult mice. Thirty exercise mice and twenty-one control mice were imaged prior to the start of the exercise regime (at 12 weeks of age) and after 4 weeks of voluntary exercise. After the second in vivo imaging session, we performed high-resolution ex vivo anatomical brain imaging to correlate the structural brain changes with functional measures of flow and vascular reserve. We found that exercise resulted in increases in the normocapnic and hypercapnic blood flow in the hippocampus. Moreover, the change in normocapnic blood flow between pre-exercise and post-exercise was positively correlated to the hippocampal structure volume following exercise. There was no overall effect of voluntary exercise on blood flow in the motor cortex. Surprisingly, the hypercapnic hippocampal blood flow when measured prior to the start of exercise was predictive of subsequent exercise activity. Moreover, exercise was found to normalize this pre-existing difference in hypercapnic blood flow between mice.This work was supported by the Canadian Institutes of Health Research Grant MOP231389