Identification of cortical arteries and veins in awake mice using two-photon microscopy.

Distinguishing arteries from veins in the cerebral cortex is critical for studying hemodynamics under pathophysiological conditions, which plays an important role in the diagnosis and treatment of various vessel-related diseases. However, due to the complexity of the cerebral vascular network, it is challenging to identify arteries and veins in vivo. Here, we demonstrate an artery-vein separation method that employs a combination of multiple scanning modes of two-photon microscopy and a custom-designed stereoscopic fixation device for mice. In this process, we propose a novel method for determining the line scanning direction, which allows us to determine the blood flow directions. The vasculature branches have been identified using an optimized z-stack scanning mode, followed by the separation of blood vessel types according to the directions of blood flow and branching patterns. Using this strategy, the penetrating arterioles and penetrating venules in awake mice could be accurately identified and the type of cerebral thrombus has been also successfully isolated without any empirical knowledge or algorithms. Our research presents a new, more accurate, and efficient method for cortical artery-vein separation in awake mice, providing a useful strategy for the application of two-photon microscopy in the study of cerebrovascular pathophysiology.

Transcranial Doppler Ultrasonography detection on cerebrovascular flow for evaluating neonatal hypoxic-ischemic encephalopathy modeling.

Objective: This study aimed to investigate the feasibility of Transcranial Doppler Ultrasonography (TCD) in evaluating neonatal hypoxic-ischemic encephalopathy (NHIE) modeling through monitoring the alteration of cerebrovascular flow in neonatal hypoxic-ischemic (HI) rats. Methods: Postnatal 7-day-old Sprague Dawley (SD) rats were divided into the control group, HI group, and hypoxia (H) group. TCD was applied to assess the changes of cerebral blood vessels, cerebrovascular flow velocity, and heart rate (HR) in sagittal and coronal sections at 1, 2, 3, and 7 days after the operation. For accuracy, cerebral infarct of rats was examined by 2,3,5-Triphenyl tetrazolium chloride (TTC) staining and Nissl staining to simultaneously verify the establishment of NHIE modeling. Results: Coronal and sagittal TCD scans revealed obvious alteration of cerebrovascular flow in main cerebral vessels. Obvious cerebrovascular back-flow was observed in anterior cerebral artery (ACA), basilar artery (BA), middle cerebral artery (MCA) of HI rats, along with accelerated cerebrovascular flows in the left internal carotid artery (ICA-L) and BA, decreased flows in right internal carotid artery (ICA-R) relative to those in the H and control groups. The alterations of cerebral blood flows in neonatal HI rats indicated successful ligation of right common carotid artery. Besides, TTC staining further validated the cerebral infarct was indeed caused due to ligation-induced insufficient blood supply. Damage to nervous tissues was also revealed by Nissl staining. Conclusion: Cerebral blood flow assessment by TCD in neonatal HI rats contributed to cerebrovascular abnormalities observed in a real-time and non-invasive way. The present study elicits the potentials to utilize TCD as an effective means for monitoring the progression of injury as well as NHIE modeling. The abnormal appearance of cerebral blood flow is also beneficial to the early warning and effective detection in clinical practice.

Mechanically manipulating glymphatic transport by ultrasound combined with microbubbles.

The glymphatic system is a perivascular fluid transport system for waste clearance. Glymphatic transport is believed to be driven by the perivascular pumping effect created by the pulsation of the arterial wall caused by the cardiac cycle. Ultrasound sonication of circulating microbubbles (MBs) in the cerebral vasculature induces volumetric expansion and contraction of MBs that push and pull on the vessel wall to generate a MB pumping effect. The objective of this study was to evaluate whether glymphatic transport can be mechanically manipulated by focused ultrasound (FUS) sonication of MBs. The glymphatic pathway in intact mouse brains was studied using intranasal administration of fluorescently labeled albumin as fluid tracers, followed by FUS sonication at a deep brain target (thalamus) in the presence of intravenously injected MBs. Intracisternal magna injection, the conventional technique used in studying glymphatic transport, was employed to provide a comparative reference. Three-dimensional confocal microscopy imaging of optically cleared brain tissue revealed that FUS sonication enhanced the transport of fluorescently labeled albumin tracer in the perivascular space (PVS) along microvessels, primarily the arterioles. We also obtained evidence of FUS-enhanced penetration of the albumin tracer from the PVS into the interstitial space. This study revealed that ultrasound combined with circulating MBs could mechanically enhance glymphatic transport in the brain.

Microvascular Environment Influences Brain Microvascular Heterogeneity: Relative Roles of Astrocytes and Oligodendrocytes for the EPCR Expression in the Brain Endothelium.

Postmortem neuropathology shows clear regional differences in many brain diseases. For example, brains from cerebral malaria (CM) patients show more hemorrhagic punctae in the brain's white matter (WM) than grey matter (GM). The underlying reason for these differential pathologies is unknown. Here, we assessed the effect of the vascular microenvironment on brain endothelial phenotype, focusing endothelial protein C receptor (EPCR). We demonstrate that the basal level of EPCR expression in cerebral microvessels is heterogeneous in the WM compared to the GM. We used in vitro brain endothelial cell cultures and showed that the upregulation of EPCR expression was associated with exposure to oligodendrocyte conditioned media (OCM) compared to astrocyte conditioned media (ACM). Our findings shed light on the origin of the heterogeneity of molecular phenotypes at the microvascular level and might help better understand the variation in pathology seen in CM and other neuropathologies associated with vasculature in various brain regions.

Computational fluid dynamics model to predict the dynamical behavior of the cerebrospinal fluid through implementation of physiological boundary conditions.

Cerebrospinal fluid (CSF) dynamics play an important role in maintaining a stable central nervous system environment and are influenced by different physiological processes. Multiple studies have investigated these processes but the impact of each of them on CSF flow is not well understood. A deeper insight into the CSF dynamics and the processes impacting them is crucial to better understand neurological disorders such as hydrocephalus, Chiari malformation, and intracranial hypertension. This study presents a 3D computational fluid dynamics (CFD) model which incorporates physiological processes as boundary conditions. CSF production and pulsatile arterial and venous volume changes are implemented as inlet boundary conditions. At the outlets, 2-element windkessel models are imposed to simulate CSF compliance and absorption. The total compliance is first tuned using a 0D model to obtain physiological pressure pulsations. Then, simulation results are compared with in vivo flow measurements in the spinal subarachnoid space (SAS) and cerebral aqueduct, and intracranial pressure values reported in the literature. Finally, the impact of the distribution of and total compliance on CSF pressures and velocities is evaluated. Without respiration effects, compliance of 0.17 ml/mmHg yielded pressure pulsations with an amplitude of 5 mmHg and an average value within the physiological range of 7-15 mmHg. Also, model flow rates were found to be in good agreement with reported values. However, when adding respiration effects, similar pressure amplitudes required an increase of compliance value to 0.51 ml/mmHg, which is within the range of 0.4-1.2 ml/mmHg measured in vivo. Moreover, altering the distribution of compliance over the four different outlets impacted the local flow, including the flow through the foramen magnum. The contribution of compliance to each outlet was directly proportional to the outflow at that outlet. Meanwhile, the value of total compliance impacted intracranial pressure. In conclusion, a computational model of the CSF has been developed that can simulate CSF pressures and velocities by incorporating boundary conditions based on physiological processes. By tuning these boundary conditions, we were able to obtain CSF pressures and flows within the physiological range.

Protein S-sulfhydration: Unraveling the prospective of hydrogen sulfide in the brain, vasculature and neurological manifestations.

Hydrogen sulfide (H2S) and hydrogen polysulfides (H2Sn) are essential regulatory signaling molecules generated by the entire body, including the central nervous system. Researchers have focused on the classical H2S signaling from the past several decades, whereas the last decade has shown the emergence of H2S-induced protein S-sulfhydration signaling as a potential therapeutic approach. Cysteine S-persulfidation is a critical paradigm of post-translational modification in the process of H2S signaling. Additionally, studies have shown the cross-relationship between S-sulfhydration and other cysteine-induced post-translational modifications, namely nitrosylation and carbonylation. In the central nervous system, S-sulfhydration is involved in the cytoprotection through various signaling pathways, viz. inflammatory response, oxidative stress, endoplasmic reticulum stress, atherosclerosis, thrombosis, and angiogenesis. Further, studies have demonstrated H2S-induced S-sulfhydration in regulating different biological processes, such as mitochondrial integrity, calcium homeostasis, blood-brain permeability, cerebral blood flow, and long-term potentiation. Thus, protein S-sulfhydration becomes a crucial regulatory molecule in cerebrovascular and neurodegenerative diseases. Herein, we first described the generation of intracellular H2S followed by the application of H2S in the regulation of cerebral blood flow and blood-brain permeability. Further, we described the involvement of S-sulfhydration in different biological and cellular functions, such as inflammatory response, mitochondrial integrity, calcium imbalance, and oxidative stress. Moreover, we highlighted the importance of S-sulfhydration in cerebrovascular and neurodegenerative diseases.

Photoacoustic imaging for in vivo quantification of alcohol-induced structural and functional changes in cerebral vasculature in high alcohol-preferring mice (HAP).

Alcohol-induced structural and functional changes were studied in vivo by photoacoustic tomography (PAT) of the cerebrovascular system in selectively bred alcohol-preferring mice. High (HAP) and low (LAP) alcohol-preferring mice are replicate lines of mice selectively bred to prefer 10% (v/v) ethanol to water and water to ethanol, respectively, in a free-access two-bottle choice scenario. A cohort of 15 singly-housed alcohol-preferring mice (five HAP mice for the experimental group, five LAP mice for the control group, and five other LAP mice set aside) were given free-access two-bottle choice 10% ethanol (v/v) and water in 50-mL graduated drinking bottles mounted on each of their cages for 4 weeks prior to PAT brain scanning. A daily log of the volume of ethanol consumed over a 24-h period was kept. At the end of the fourth week, blood samples were collected from the HAP mice and blood ethanol concentrations (BECs) were measured to ascertain their levels of ethanol intoxication. The mice were then grouped into five weight-matched pairs of HAP and LAP for comparison purposes, and noninvasive in vivo PAT imaging was performed on each weight-matched pair. To mimic a binge drinking paradigm, mice were rearranged into four weight-matched groups of three animals each: an HAP mouse and two LAP mice. For each group, one HAP mouse and one LAP mouse received a 20% ethanol solution via intraperitoneal (i.p.) injection after 24 h of ethanol abstinence, in weight-based doses of 3 g/kg prior to imaging, while the last LAP mouse received a sham i.p. injection. PAT images of the brain were collected for 30 min thereafter. Cerebral vascular diameters for selected vessels of interest were extracted from the PAT images and compared between HAP mice and LAP mice. For the binge scenario, changes in vessel diameter and hemoglobin oxygen saturation were extracted from PAT images and studied over a 30-min duration. Vascular diameter was significantly smaller in HAP mice compared to LAP mice in weight-matched pairs. Hemoglobin-oxygen saturation and vessel diameter dropped more quickly in LAP mice than in HAP mice following a 20% ethanol i.p. injection (3 g/kg), with a 32% reduction in cerebrovascular diameter in a 30-min period. This study demonstrates the effectiveness of PAT in alcohol addiction imaging and diagnosis, and its feasibility in studying alcohol-induced changes in vascular structure and perfusion. It also adds to other bodies of evidence to suggest that the effects of binge drinking are more adverse in occasional drinkers than habitual drinkers.

Inflammation: A Mediator Between Hypertension and Neurodegenerative Diseases.

Hypertension is the most prevalent and modifiable risk factor for stroke, vascular cognitive impairment, and Alzheimer's disease. However, the mechanistic link between hypertension and neurodegenerative diseases remains to be understood. Recent evidence indicates that inflammation is a common pathophysiological trait for both hypertension and neurodegenerative diseases. Low-grade chronic inflammation at the systemic and central nervous system levels is now recognized to contribute to the physiopathology of hypertension. This review speculates that inflammation represents a mediator between hypertension and neurodegenerative diseases, either by a decrease in cerebral blood flow or a disruption of the blood-brain barrier which will, in turn, let inflammatory cells and neurotoxic molecules enter the brain parenchyma. This may impact brain functions including cognition and contribute to neurodegenerative diseases. This review will thus discuss the relationship between hypertension, systemic inflammation, cerebrovascular functions, neuroinflammation, and brain dysfunctions. The potential clinical future of immunotherapies against hypertension and associated cerebrovascular risks will also be presented.

Microscope integrated optical coherence tomography of a cerebral arachnoid cyst: A new technique to increase intraoperative security.

PURPOSE: This technical note illustrates microscope integrated optical coherence tomography (iOCT) as an imaging technique to delineate concealed micro anatomical structures not displayable by conventional intraoperative imaging methods in the context of a cerebral arachnoid cyst. METHODS: iOCT was used for the first time to scan a cerebral arachnoid cyst in vivo. Scanning sites were defined at the outer membrane of the arachnoid cyst, the inner membrane at the temporal cortex as well as at the fenestration site to the basal cisterns - a point out of reach and resolution for conventional intraoperative imaging methods like e. g. ultrasound or neuroendoscopy. RESULTS: iOCT was feasible during microsurgical fenestration of an arachnoid cyst. A clear delineation of the arachnoid cyst membrane was possible. The differentiation of the arachnoid cyst membrane and underlying arachnoid barrier cell membrane was possible. Trans cystic scanning at the temporal cortex could delineate the content of the subarachnoid space like subarachnoid blood vessels, trabecular sytem and vessel wall morphology of a M4 middle cerebral artery branch. Scanning of the inner membrane of the arachnoid cyst at site of fenestration to the basal cisterns excluded underlying micro anatomical structures. CONCLUSION: This case demonstrates that iOCT achieved to delineate concealed micro anatomical structures which are occult to conventional intraoperative imaging methods. Further studies are necessary to value iOCT as a tool to improve intraoperative security.

Vascular α1A Adrenergic Receptors as a Potential Therapeutic Target for IPAD in Alzheimer's Disease.

Drainage of interstitial fluid from the brain occurs via the intramural periarterial drainage (IPAD) pathways along the basement membranes of cerebral capillaries and arteries against the direction of blood flow into the brain. The cerebrovascular smooth muscle cells (SMCs) provide the motive force for driving IPAD, and their decrease in function may explain the deposition of amyloid-beta as cerebral amyloid angiopathy (CAA), a key feature of Alzheimer's disease. The α-adrenoceptor subtype α1A is abundant in the brain, but its distribution in the cerebral vessels is unclear. We analysed cultured human cerebrovascular SMCs and young, old and CAA human brains for (a) the presence of α1A receptor and (b) the distribution of the α1A receptor within the cerebral vessels. The α1A receptor was present on the wall of cerebrovascular SMCs. No significant changes were observed in the vascular expression of the α1A-adrenergic receptor in young, old and CAA cases. The pattern of vascular staining appeared less punctate and more diffuse with ageing and CAA. Our results show that the α1A-adrenergic receptor is preserved in cerebral vessels with ageing and in CAA and is expressed on cerebrovascular smooth muscle cells, suggesting that vascular adrenergic receptors may hold potential for therapeutic targeting of IPAD.

Preparation and Culturing of Human Primary Vascular Cells.

Cerebral amyloid angiopathy (CAA) results from amyloid accumulation within arteries of the cerebral cortex and leptomeninges. This condition is age-related, especially prevalent in Alzheimer's disease (AD), and the main feature of certain hereditary disorders (i.e., HCHWA-I). The vascular smooth muscle cells (VSMCs) appear to play a vital role in the development of CAA, which makes them well suited as an experimental model to study the disease and screen for possible remedies. We describe two different methods for isolating and culturing human VSMCs: First, using the human umbilical cord as an easy source of robust cells, and secondly, using brain tissue that provides the proper cerebral VSMCs, but is more problematic to work with. The umbilical cord also provides human umbilical vascular endothelial cells (HUVEC), useful primary cells for vascular research. Finally, the maintenance, preservation, and characterization of the isolated vascular cells are described.

The influence of the carotid baroreflex on dynamic regulation of cerebral blood flow and cerebral tissue oxygenation in humans at rest and during exercise.

PURPOSE: This preliminary study tested the hypothesis that the carotid baroreflex (CBR) mediated sympathoexcitation regulates cerebral blood flow (CBF) at rest and during dynamic exercise. METHODS: In seven healthy subjects (26 ± 1 years), oscillatory neck pressure (NP) stimuli of + 40 mmHg were applied to the carotid baroreceptors at a pre-determined frequency of 0.1 Hz at rest, low (10 ± 1W), and heavy (30 ± 3W) exercise workloads (WLs) without (control) and with α - 1 adrenoreceptor blockade (prazosin). Spectral power analysis of the mean arterial blood pressure (MAP), mean middle cerebral artery blood velocity (MCAV), and cerebral tissue oxygenation index (ScO2) in the low-frequency range (0.07-0.20 Hz) was estimated to examine NP stimuli responses. RESULTS: From rest to heavy exercise, WLs resulted in a greater than three-fold increase in MCAV power (42 ± 23.8-145.2 ± 78, p < 0.01) and an almost three-fold increase in ScO2 power (0.51 ± 0.3-1.53 ± 0.8, p = 0.01), even though there were no changes in MAP power (from 24.5 ± 21 to 22.9 ± 11.9) with NP stimuli. With prazosin, the overall MAP (p = 0.0017), MCAV (p = 0.019), and ScO2 (p = 0.049) power was blunted regardless of the exercise conditions. Prazosin blockade resulted in increases in the Tf gain index between MAP and MCAV compared to the control (p = 0.03). CONCLUSION: CBR-mediated changes in sympathetic activity contribute to dynamic regulation of the cerebral vasculature and CBF at rest and during dynamic exercise in humans.

Standardization of the technique of silicon injection of human cadaveric heads for opacification of cerebral vasculature in Indian conditions.

A surgeon's understanding of the surgical anatomy can be greatly enhanced by the dissection of preserved cadaveric specimens. A reliable and inexpensive biological model for testing and standardization of dye injection concentrations is proposed utilizing the goat's head as a biological model. The first phase was concerned with standardization of the dye by titrating its concentration and injecting various amounts into cerebral vessels of a goat's head until an optimal concentration had been ascertained. In the second phase, this optimum concentration of the dye was injected into four human cadaveric heads following the same technique standardized using the goat's head. Upon dissecting the four cadaveric human heads which were injected with silicon dyes and preserved in 10% formalin, the vessels were all well-opacified and the brain was of near normal consistency and good for dissection, without showing any features of putrefaction. The goat model, having similar color, texture, and the handling as the cadaveric head, offers an opportunity to test indigenously manufactured polymerizing dyes in the future. This biological model, therefore, has the potential to considerably reduce the cost of cadaver preparation.

[Changes in the brain vascular bed associated with sudden death of young subjects]. / Izmeneniia sosudistogo rusla golovnogo mozga pri vnezapnoi smerti lits molodogo vozrasta.

This article presents an overview of the literature publications concerning pathological changes in the cerebral blood vessels and the factors underlying the development of hemorrhagic complications leading to sudden death of young people. The special emphasis is placed on the most important causes behind the changes in the vascular wall (including the congenital ones) responsible for the high risk of rupture of the intracerebral vessels associated with the development of hemorrhagic complications.

Cerebrovascular dysfunction following subfailure axial stretch.

Cerebral blood vessels are vital to maintaining the health of the brain. Traumatic brain injury (TBI) commonly results in autoregulatory dysfunction and associated failure of cerebral vessels to maintain homeostasis in the brain. While post-injury changes to brain biochemistry are known to contribute to this dysfunction, tissue deformation may also directly alter vascular smooth muscle cell (SMC) function. As a first step toward understanding stretch-induced dysfunction, this study investigates the effect of overstretch on the contractile behavior of SMCs in middle cerebral arteries (MCAs). We hypothesized that vessel function is altered above a threshold of stretch and strain rate. Twenty-four MCAs from Sprague Dawley rats were tested. Following development of basal SMC tone, vessels were subjected to increasing levels of isosmotic extracellular potassium (K+). Samples were then subjected to an axial overstretch of either 1.2*λIV or 1.3*λIV at strain rates of 0.2 or 20s-1. Following overstretch, SMC contractile behavior was measured again, both immediately and 60min after overstretch. Control vessels were subjected to the same protocol but without overstretch. SMC contractile behavior was characterized using both percent contraction (%C) relative to the fully dilated inner diameter and the K+ dose required to evoke the half maximal contractile response (EC50). Control vessels exhibited increased sensitivity to K+ in successive characterization tests, so all effects were quantified relative to the time-matched control response. Samples exhibited the typical biphasic response to extracellular K+, dilating and contracting in response to small and large K+ concentrations, respectively. As hypothesized, axial overstretch altered SMC contractile behavior, as seen in a decrease in %C for sub-maximal contractile K+ doses (p<0.05) and an increase in EC50 (p<0.01), but only for the test group stretched rapidly to 1.3*λIV. While the change in %C was only significantly different immediately after overstretch, the change to EC50 persisted for 60min. These results indicate that deformation can alter SMC contractile behavior and thus potentially play a role in cerebrovascular autoregulatory dysfunction independent of the pathological chemical environment in the brain post-TBI.

A simple approach for non-invasive transcranial optical vascular imaging (nTOVI).

In vivo imaging of cerebral vasculature is highly vital for clinicians and medical researchers alike. For a number of years non-invasive optical-based imaging of brain vascular network by using standard fluorescence probes has been considered as impossible. In the current paper controverting this paradigm, we present a robust non-invasive optical-based imaging approach that allows visualize major cerebral vessels at the high temporal and spatial resolution. The developed technique is simple to use, utilizes standard fluorescent dyes, inexpensive micro-imaging and computation procedures. The ability to clearly visualize middle cerebral artery and other major vessels of brain vascular network, as well as the measurements of dynamics of blood flow are presented. The developed imaging approach has a great potential in neuroimaging and can significantly expand the capabilities of preclinical functional studies of brain and notably contribute for analysis of cerebral blood circulation in disorder models. An example of 1 × 1.5 cm color-coded image of brain blood vessels of mouse obtained in vivo by transcranial optical vascular imaging (TOVI) approach through the intact cranium.

Physical exercise-induced protection on ischemic cardiovascular and cerebrovascular diseases.

Physical exercise is any bodily activity to enhance or maintain physical fitness and overall health and wellness. A series of associated studies have demonstrated that physical exercise could alleviate the infarct volume, increase the collateral circulation, promote endothelial progenitor cells, improve cerebral blood flow after cardiovascular and cerebrovascular diseases. In this review, we summed up the protective effects of physical exercise on cerebral blood flow (CBF), vascular endothelium, vascular vasodilation, endothelial progenitor cells and collateral circulation. An awareness of the exercise intervention benefits for cardiovascular and cerebrovascular diseases may encourage more patients with cerebral infarction and myocardial infarction and people with high risk factors to accept exercise interventions for the prevention and treatment of cardiovascular and cerebrovascular diseases.

Physical exercise training and neurovascular unit in ischemic stroke.

Physical exercise could exert a neuroprotective effect in both clinical studies and animal experiments. A series of related studies have indicated that physical exercise could reduce infarct volume, alleviate neurological deficits, decrease blood-brain barrier dysfunction, promote angiogenesis in cerebral vascular system and increase the survival rate after ischemic stroke. In this review, we summarized the protective effects of physical exercise on neurovascular unit (NVU), including neurons, astrocytes, pericytes and the extracellular matrix. Furthermore, it was demonstrated that exercise training could decrease the blood-brain barrier dysfunction and promote angiogenesis in cerebral vascular system. An awareness of the exercise intervention benefits pre- and post stroke may lead more stroke patients and people with high-risk factors to accept exercise therapy for the prevention and treatment of stroke.

Genomic and non-genomic regulation of PGC1 isoforms by estrogen to increase cerebral vascular mitochondrial biogenesis and reactive oxygen species protection.

We previously found that estrogen exerts a novel protective effect on mitochondria in brain vasculature. Here we demonstrate in rat cerebral blood vessels that 17ß-estradiol (estrogen), both in vivo and ex vivo, affects key transcriptional coactivators responsible for mitochondrial regulation. Treatment of ovariectomized rats with estrogen in vivo lowered mRNA levels of peroxisome proliferator-activated receptor-γ coactivator-1 alpha (PGC-1α) but increased levels of the other PGC-1 isoforms: PGC-1ß and PGC-1 related coactivator (PRC). In vessels ex vivo, estrogen decreased protein levels of PGC-1α via activation of phosphatidylinositol 3-kinase (PI3K). Estrogen treatment also increased phosphorylation of forkhead transcription factor, FoxO1, a known pathway for PGC-1α downregulation. In contrast to the decrease in PGC-1α, estrogen increased protein levels of nuclear respiratory factor 1, a known PGC target and mediator of mitochondrial biogenesis. The latter effect of estrogen was independent of PI3K, suggesting a separate mechanism consistent with increased expression of PGC-1ß and PRC. We demonstrated increased mitochondrial biogenesis following estrogen treatment in vivo; cerebrovascular levels of mitochondrial transcription factor A and electron transport chain subunits as well as the mitochondrial/nuclear DNA ratio were increased. We examined a downstream target of PGC-1ß, glutamate-cysteine ligase (GCL), the rate-limiting enzyme for glutathione synthesis. In vivo estrogen increased protein levels of both GCL subunits and total glutathione levels. Together these data show estrogen differentially regulates PGC-1 isoforms in brain vasculature, underscoring the importance of these coactivators in adapting mitochondria in specific tissues. By upregulating PGC-1ß and/or PRC, estrogen appears to enhance mitochondrial biogenesis, function and reactive oxygen species protection.