Role of Mitochondria in Cerebral Vascular Function: Energy Production, Cellular Protection, and Regulation of Vascular Tone

Abstract

Mitochondria not only produce energy in the form of ATP to support the activities of cells comprising the neurovascular unit, but mitochondrial events, such as depolarization and/or ROS release, also initiate signaling events which protect the endothelium and neurons against lethal stresses via pre-/postconditioning as well as promote changes in cerebral vascular tone. Mitochondrial depolarization in vascular smooth muscle (VSM), via pharmacological activation of the ATP-dependent potassium channels on the inner mitochondrial membrane (mitoK_ATP channels), leads to vasorelaxation through generation of calcium sparks by the sarcoplasmic reticulum and subsequent downstream signaling mechanisms. Increased release of ROS by mitochondria has similar effects. Relaxation of VSM can also be indirectly achieved via actions of nitric oxide (NO) and other vasoactive agents produced by endothelium, perivascular and parenchymal nerves, and astroglia following mitochondrial activation. Additionally, NO production following mitochondrial activation is involved in neuronal preconditioning. Cerebral arteries from female rats have greater mitochondrial mass and respiration and enhanced cerebral arterial dilation to mitochondrial activators. Preexisting chronic conditions such as insulin resistance and/or diabetes impair mitoK_ATP channel relaxation of cerebral arteries and preconditioning. Surprisingly, mitoK_ATP channel function after transient ischemia appears to be retained in the endothelium of large cerebral arteries despite generalized cerebral vascular dysfunction. Thus, mitochondrial mechanisms may represent the elusive signaling link between metabolic rate and blood flow as well as mediators of vascular change according to physiological status. Mitochondrial mechanisms are an important, but underutilized target for improving vascular function and decreasing brain injury in stroke patients.

Introduction

The neurovascular unit represents the functional integration of cerebral vascular cells: endothelium, vascular smooth muscle (VSM), pericytes, perivascular neurons, and the parenchymal cells: neurons, astroglia, oligodendrites, and microglia. A primary purpose of the neurovascular unit is to couple brain metabolic demand with glucose and oxygen delivery via the blood as well as to adjust cerebral vascular resistance appropriately to systemic influences, such as blood gases and pH, circulating hormones and neurotransmitters, blood pressure, and shear stress. Similar functional arrangements, but with different vascular and tissue characteristics, occur in other regional circulations. Many excellent reviews have discussed various influences on the regulation of the cerebral vasculature (11,13,19,32,45,54–57,76,77,80). Unique aspects of the cerebral circulation include an important role of large arteries in altering vascular resistance, extensive perivascular innervation, and restricted permeability across the cerebral vascular endothelium.

Recent evidence indicates that mitochondria are important elements in cerebral vascular function during health and disease (14, 26, 46, 48, 85–87, 131, 134), but the extent of knowledge is limited. The purpose of this review is to explore the evidence for an important role of mitochondrial-based mechanisms on the function of the cerebral circulation. This area of investigation is relatively new and additional work is needed not only on the cerebral circulation but also on other regional circulations. This review will focus on mitochondrial aspects of energy production, cellular protection, and regulation of vascular tone in cerebral vascular cells as well as in other cell types of the neurovascular unit. This discussion is limited to the systemic circulation because mitochondrial mechanisms as well as vascular control mechanisms in the pulmonary circulation are very different (46).

Several excellent reviews on mitochondria in the peripheral endothelium (95,146) or VSM (22,125) emphasize the importance of this emerging research area. However, little attention has been given to the integration of mitochondrial-generated signals in various cells within the neurovascular unit or other regional circulations. This review is unique because it explores the commonalities of the shared mitochondria-initiated signaling pathways for preconditioning, energy production, and vasoreactivity. We will also present data establishing that mitochondrial dynamics are affected by sex, which has been a neglected area of research (131). Moreover, we will show that even mild metabolic stress, such as occurs with insulin resistance (IR), a component of the metabolic syndrome, can dramatically affect mitochondrial related events in the cerebral vasculature (85,86). Lastly, we will present novel findings which indicate that mitochondrial based dilator pathways, located largely in the endothelium, are intact following ischemic stress, and that targeting these mechanisms may benefit patients following stroke (132).

Mitochondrial Morphology

Mitochondria are double-membrane organelles which generate energy in the form of ATP which subsequently promotes the activities of cells comprising the neurovascular unit (65,81). Important structural features of mitochondria are: (i) a relatively permeable outer membrane, containing among other entities, the voltage-gated anion channel (VDAC); (ii) a relatively impermeable inner membrane; (iii) the intermembrane space; (iv) extensions of the inner membrane called cristae; and (v) the intracristae spaces referred to as the matrix (81,151). Each anatomical component has a specific function, which is essential for the optimal production and transfer of energy from the mitochondria into the cytosol. The Krebs cycle, located in the matrix, and Complexes IV, associated with the electron transport chain, are embedded in the relatively impermeable inner mitochondrial membrane. The mitochondrial membrane potential (Δψm) across the inner membrane, which is normally maintained at −120 to −180 mV, provides the proton difference used to drive the synthesis of ATP by the electron-transport chain. Several diagnostic and therapeutic approaches take advantage of this negative membrane potential to target membrane permeable, positively charged agents specific to the mitochondria (39,41,67,84,121). Detailed descriptions of ATP production by nonmitochondrial glycolysis as well as the mitochondrial electron transport chain are widely available.

Mitochondria possess their own DNA, distinct from nuclear DNA, and also contain protein synthesizing machinery which produces approximately one-half of their proteins, whereas the other half are encoded by nuclear DNA and imported into mitochondria via several different transport mechanisms. The cytochrome c oxidase complex (Complex IV), the final component of the electron-transport chain localized to the inner membrane, is a good example of the dual origins of the proteins present in mitochondria. Ten subunits are nuclear in origin and are transported into the mitochondria and three are synthesized within the mitochondria (81,103).

Mitochondria are dynamic organelles, not static entities. Mitochondria can undergo replication, fission and fusion, move from one location to another within cells, and form networks with other mitochondria or cellular structures to increase efficiency of ATP production as well as providing for intracellular signaling in response to physiological and pathological stimuli (30, 34, 46, 47, 49, 145, 147, 149). Typical morphological characteristics of mitochondria in cells of the neurovascular unit during a variety of conditions are presented in Figures 1 to 9. For purposes of illustration, we have chosen representative images from different cell types of the neurovascular unit, which demonstrate the variety of mitochondrial numbers, morphology, locations, and relationships to other cellular structures. In particular, the mitochondrial features in cerebral arteries and brain parenchyma are similar to those published previously (14,116,131). Additionally, we have published examples of morphological features of isolated brain, liver and cardiac mitochondria, mitochondria in cultured and intact cortical neurons, mitochondria in cultured cerebral vascular endothelium as well as mitochondria in intact arteries from male and female rats and hearts from normal and IR rats (14,87,101,116,131,145). Although individual mitochondria form extensive networks among themselves within cultured cerebral endothelial cells (14), it is not clear whether such interconnections occur under normal conditions in endothelium in vivo. However, the mitochondrial outer membranes in VSM are often connected or closely jux-tapositioned with themselves as well as with the sarcoplasmic reticulum (SR) (Figs. 1–3). The sarcoplasmic reticula are cellular structures involved in calcium storage and release; the complex interplay among mitochondrial, SR, and cytosolic calcium has been extensively described (21,30,44,137,148). The clustering of large numbers of mitochondria with the SR might impair mitochondrial movement seen in other cell types during normal conditions. However, mitochondria in VSM have been reported to be mobile during pathological conditions when this cell type is undergoing migration and remodeling (22).

Figure 1.

Electron microscopy section of a small branch of the MCA representative of 10- to 12-week-old, male Sprague Dawley (SD) rats showing differences in size, morphology, and relationship to other cellular structures of mitochondria in VSM and endothelium. Mitochondria in VSM form large fields with interspersed SR, whereas mitochondria in endothelium seem to be present singly. Rats were euthanized with anesthesia and perfused with a PBS solution containing 2% glutaraldehyde and 3% formaldehyde. Arteries were removed and kept in the perfusion solution for 1 h and post fixed in 1% osmium tetroxide and embedded in Spurr’s resin. Ultrathin sections (80–90 nm) were mounted on formvar-coated copper grids (200 mesh), air dried, and stained with uranyl acetate and lead citrate (at 7 and 7 min, respectively). The sections were put on grids and viewed at a magnification of 11,000x using a FEI Tecnai Bio Twin 120 keV TEM with a digital imaging setup (Wake Forest University Health Sciences, Winston-Salem, NC). M, mitochondrion; SR, sarcoplasmic reticulum, IEL, internal elastic lamina, VSM, vascular smooth muscle. Magnification is 11,000x.

Figure 9.

Electron microscopy section showing the VSM of a cerebral artery representative of 10- to 12-week-old, male rats showing damage to mitochondria in VSM at 48 h of reperfusion following 90 min of transient MCA occlusion. Mitochondria appear to be swollen and/or have lost the typical morphology including uniform cristae. Mitochondria in VSM cells following ischemia can have diverse features ranging from a normal appearance or show limited or extensive damage. Nonetheless, dilator responses to sodium nitroprusside, a VSM specific stimulus, are intact following ischemia. Magnification 11,000x.

Figure 3.

Higher magnification of mitochondrial connections in a VSM cell representative of 10-to 12-week-old, female SD rats. Numerous apparent contacts between mitochondria (examples shown by white arrows) as well as close approximation of mitochondria and SR. Magnification is 23,000x.

Pericytes commonly extend longitudinally down the length of capillaries, but can occasionally encircle individual capillaries (109). Several studies have implicated an important role of pericytes in the regulation of cerebral microcirculation but the issue is controversial (32, 56). We have observed that pericytes often have very large mitochondria relative to their size or compared to adjacent endothelium (Fig. 4). Astroglial endfeet (Figs. 4–6) surround cerebral blood vessels within the parenchyma (109). We find that in addition to round and oblong mitochondria, astroglia often contain elongated mitochondria as well as unusually shaped mitochondria. However, regardless of shape, all of these astroglial mitochondria are seen located close to vascular cells in the parenchyma or on the pial surface (Figs. 4–6). Pial arteries, an important site of resistance in the cerebral circulation, are normally located on a layer of astroglia (glia limitans) on the cortical surface which contain numerous mitochondria (Figs. 6). The adventitial surface of large and pial arteries, composed of connective tissue cells as well as perivascular nerves, also contain large numbers of mitochondria (Figs. 6 and 7). Parenchymal neurons in situ and in culture are heavily invested with mitochondria in accordance with their high metabolic needs (50, 145; Figs. 4 and 6). Mitochondria also are located at junctions of perivascular nerve terminals with VSM (Fig. 8). Pathological conditions, such as acute ischemia, can damage mitochondria to an extent that morphological changes in mitochondria are obvious and widespread (Fig. 9). In our experience, different VSM cells from the same artery as well as from arteries of rats similarly exposed to transient brain ischemia show variable responses where mitochondrial damage ranges from very extensive to scattered evidence of damage to no apparent mitochondrial injury. Nonetheless, in the broader sense, mitochondrial changes following ischemia or anoxic stress in the neurovascular unit are complex and specific to cell type (132,135,145). In summary, large numbers of mitochondria are present within the cells composing the cerebral blood vessels and the adjacent neurons and astroglia, thus establishing the potential for these organelles to be key components of cerebral vascular function during health and disease as well as therapeutic targets.

Figure 4.

Electron microscopy section of a microvessel in brain parenchyma representative of 10- to 12-week-old, female SD rats showing a surrounding pericyte with a large mitochondrion as well as mitochondria in astrocytic endfeet. The pericytes and especially astroglial endfeet surround the parenchymal blood vessels and both cell types contain pronounced mitochondria. Magnification is 11,000x. M = mitochondrion, * = astroglial endfoot.

Figure 6.

Electron microscopy section of a pial artery representative of 10- to 12-week-old, male Zucker lean rats showing multiple mitochondria on glia limitans comprising the surface of cerebral cortex as well as in cell layers in adventitial connective tissues and nerves. Fields of mitochondria in astrocytes are shown in the white oval areas and the dashed rectangle corresponds to the area of the section enlarged in Figure 7. Magnification 1900x.

Figure 7.

Higher magnification of indicated section of Figure 4 showing numerous mitochondria in perivascular tissue. White oval shows mitochondrial field in VSM and black oval shows mitochondrial in adventitia. M, mitochondrion, VSM, vascular smooth muscle. Magnification 11,000x.

Figure 8.

Electron microscopy section of a MCA representative of 10- to 12-week-old SD rats showing perivascular innervation. Mitochondria are prominent features in nerve terminals. Myelinated and nonmyelinated nerves are seen associated with the adventitia. M, mitochondrion. Magnification is 30,000x.

Although it has been suggested that mitochondria contain a variant of nitric oxide synthase (NOS) in normal cells, and thus could produce NO via this enzymatic pathway, there is significant evidence against this view (98–100). Nonetheless, NO arising from nonmitochondrial sources, such as endothelial cells, VSM, astroglia, pericytes, and neurons (88, 89) can affect the functioning of the electron transport chain by reducing mitochondrial oxygen consumption and ATP production (132) and could combine with superoxide anion to form peroxynitrite (54,58,145). We have shown that peroxynitrite is a potent activator of mitochondria (101). On the other hand, we have shown that NO production from cytosolic NOS occurs due to NOS phosphorylation and/or increased cytosolic calcium in endothelial cells and neurons following pharmacological activation of mitochondria (88, 89). NO appears to have a restraining effect on mitochondrial oxygen consumption in intact cerebral arteries (132).

Mitochondrial Energetics and Reactive Oxygen Species Production

Mitochondria are the most prominent consumers of oxygen by cells and are the major site of ATP production by cerebral arteries (113,132). The rate of maximal mitochondrial oxygen consumption is positively correlated with mitochondrial protein mass, but this relationship can vary depending upon the sizes and status of the mitochondria. In turn, brain blood flow is directly correlated with oxygen consumption (2,17,18). In addition to ATP production, mitochondria in neurons, astroglia, VSM, and cerebral vascular endothelium are constant producers of reactive oxygen species (ROS) in the form of superoxide anion via the electron transport chain (15,64,78,88,127). Mitochondrial-derived ROS in healthy cells appear to be important, if not essential, signaling agents involved in the maintenance of basal cell functions. Superoxide anion is produced from the respiratory complexes within the matrix and usually leaves the matrix (71,72,106), after enzymatic conversion by manganese superoxide dismutase (MnSOD) to hydrogen peroxide, through aquaporin-like channels in the inner mitochondrial membrane (8, 20, 107). Manganese SOD, which has a high capacity for converting superoxide ion to hydrogen peroxide and water, rapidly eliminates superoxide anion as it is formed in the matrix. Hydrogen peroxide in the intermembrane space, or superoxide anion directly released into the intermembrane space by Complex III under certain conditions, can easily transverse the mitochondrial outer membrane, via VDACs or other pathways, into the cytosol (10,71,118). Under conditions such as shear stress, a physical distortion of the relationship between the two mitochondrial membranes in the endothelium leads to augmented ROS production and release by mitochondria resulting in coronary artery dilation via a hydrogen peroxide-based mechanism (146, 149). Recent evidence indicates that protein kinases in the intermembrane space are targets of mitochondrial-generated ROS and thus may initiate signaling cascades within mitochondria as well as within the cytosol (122).

The primary sites of superoxide anion production and release are Complex I (NADH-ubiquinone oxidoreductase), Complex II (succinate dehydrogenase, SDH), and Complex III (ubiquinol-cyctochrome c oxidoreductase). Complexes I and II accept electrons from NADH + H⁺ and FADH₂, respectively, which are transferred to Complex III and finally to Complex IV (cytochrome c oxidase), where the final electron acceptor is oxygen and the final product is water (8, 65, 72). Superoxide anion from these three complexes is released into the matrix. A number of metabolic poisons are available which inhibit one or more of the respiratory chain complexes: rotenone: Complex I; 3-nitropropionic acid (3-NPA): Complex II; antimycin A: Complex III; cyanide: Complex IV; and oligomycin: Complex V. The result of inhibition via these metabolic poisons, especially of Complexes I-III, is enhanced ROS release. Some of these agents as well as FCCP are used in the Seahorse Bioscience Analyzer to characterize the roles of mitochondrial and nonmitochondrial activities in cellular oxygen consumption.

Although the continuous release of ROS from mitochondria during normal conditions appears to play a necessary role in the maintenance of basal cellular function, transiently elevated ROS levels can promote selective protein synthesis, preconditioning, and changes in vascular tone. However, even modest, chronically elevated mitochondrial ROS production can lead to cellular dysfunction. We have shown that a mutation of the inner mitochondrial membrane peptidase 2-like (Immp2l) gene leads to chronically enhanced ROS release by mitochondria, subsequently causing reduced vascular dilation to the administration of carbachol in mesenteric arteries (106). Similarly, chronically elevated mitochondrial ROS production due to the metabolic syndrome impairs cerebral vascular function via effects on plasmalemmal as well as mitochondrial K_ATP channels through multiple mechanisms (5,6,51–53,87). It also has been reported that a genetic deficiency of MnSOD in mice increases basal superoxide levels in cerebral arteries and aorta (10). Moreover, Pung et al. have shown that chronic inhibition of Complex I by rotenone blocks ischemia induced collateral artery growth in the coronary circulation via activation of adenosine monophosphate-activated kinase and the subsequent inhibition of mechanistic target of rapamycin (mTOR) and p70 ribosomal S6 kinase (126). Higher levels of cellular ROS, such as occurs during injury and disease, can lead to cell death via both mitochondrial- and nonmitochondrial-mediated pathways, especially in metabolically compromised conditions (6,23,29,38,93,138).

Although production of ROS by mitochondria appears to be an independent process under most conditions, recent reports indicate that ROS production by mitochondria can promote ROS production by the extramitochondrial NADPH oxidase system, or vice versa, via a positive feedback system (23,31,37,134,138). Thus, the interaction between the mitochondria and the cytosolic NADPH oxidase axis leads to cellular damage due to excessive production of ROS by a “ROS induced ROS” mechanism. Mitochondria located near the inner plasma membrane surface appear to mediate this response (24). The best example of this interaction occurs with the exposure of vascular and nonvascular cells to angiotensin II (24,31,37,38,134). Hypertension can cause increased mitochondrial-mediated ROS stress in the cerebral vasculature (38). Furthermore, aging can exacerbate pressure-induced mitochondrial ROS production in mouse cerebral arteries (29,138). Genetically induced deficiency of MnSOD also leads to cerebral vascular endothelial dysfunction, especially in aged mice to angiotensin II (27,117). We speculate that increased production of mitochondrial ROS will lead to deterioration of cerebral vascular function and associated cognitive function in aging people. Chalmers et al. (23) recently have shown that hypertension also alters the architecture of mitochondria in arteries.

Mitochondrial Depolarization and Related Signaling Events

Mitochondrial depolarization is accompanied by superoxide anion generation during many physiological and pathological conditions and also when inhibitors of mitochondrial complexes are administered. Thus, it was assumed for many years that mitochondrial depolarization always leads to increased superoxide anion production. Our findings, however, demonstrated for the first time that mitochondrial depolarization and enhanced mitochondrial ROS production are not mandatorily linked events in cells. In isolated brain mitochondria, cultured neurons, astroglia and cerebral vascular endothelium, and intact cerebral vascular endothelium and VSM, the agent BMS-191095 causes mitochondrial depolarization without ROS production and promotes diverse cellular responses such as pre-/post-conditioning and VSM relaxation; thus, mitochondrial depolarization alone is sufficient to activate distinct cellular signaling pathways (12,15,64,86,88,89). Our findings with BMS-191095 are supported by results from other laboratories (126,143,144) and by other experimental approaches in our laboratory (41).

The previous concept linking mitochondrial membrane potential and ROS production/release arose from experiments using inhibitors of the electron transport chain, which reduced the ATP levels necessary to maintain the very negative transmembrane potential of mitochondria. However, the dissociation between mitochondrial membrane potential and ROS release is not absolute, but probably occurs within a limited range of depolarization and may be cell type specific or affected by sex (131). The relative dissociation of mitochondrial membrane potential from ROS production and release, with the subsequent induction of different signaling pathways, underscores the versatility of mitochondria to initiate appropriate and selective cellular responses to varied stimuli.

The most effective and reproducible approach that we have found to depolarize mitochondria is by activating mitoK_ATP channels on the inner mitochondrial membrane with drugs such as BMS-191095 or diazoxide (14,28,55). The physical structure of the mitoK_ATP channel is not known with certainty but appears to differ substantially from the previously described plasmaK_ATP channels (1,59,60,102,129, 133). Several reports have provided evidence that the renal outer medullary potassium channel is a component of the K⁺ channel of the cardiac mitoK_ATP channel (59,129). SDH might also be involved in either the assembly or function of the mitoK_ATP channel (122). The pharmacology of the mitoK_ATP channel function is well validated; however, additional studies are needed to elucidate the complete structure of the mitoK_ATP channels. The activity of the mitoK_ATP channel is normally affected by endogenous factors such as the ADP/ATP ratio (1) as well as the ambient levels of peroxynitrite (101, 102), superoxide anion (101,102,124), and cytosolic protein kinase C epsilon (PKCe) (124). Other, yet unidentified, physiological and pathological factors likely will be found to directly or indirectly activate or block the activity of the mitoK_ATP channel. Mitochondria depolarize under normal conditions (121) and naturally occurring substances, such as oxytocin depolarize mitochondria in myometrial cells by either direct or indirect mechanisms (68).

The consequences to the cell of activating mitoK_ATP channels as opposed to plasmaK_ATP channels are quite different. The mitochondrial matrix has a very negative potential and opening of the mitoK_ATP channels allows potassium to enter the matrix because the K⁺ gradient makes the potential less negative, thereby “depolarizing” the mitochondrion. In contrast, opening the plasmaK_ATP channels allows potassium to exit the cytosol into the extracellular space making the cytosol more negative and thereby “hyperpolarizing” the cell. Nonetheless, our studies have shown that the function of K_ATP channels in either cellular location are negatively affected by chronic, modest levels of ROS in IR (52,85,87).

Isolated mitochondria and mitochondria in situ in cultured endothelial cells, astroglia, and neurons, and also in intact endothelial and VSM cells in isolated cerebral arteries depolarize in a dose-dependent manner to selective mitoK_ATP channel openers such as diazoxide and BMS-191095 (9,14–16,55,63,64,86,88,89). The classical K_ATP channel antagonist, glibenclamide, as well as 5-hydroxydecanoic acid, which needs to be metabolized by cells before becoming active (82), block the actions of diazoxide or BMS-191095 (15,64). Diazoxide, a drug previously used in the treatment of acute hypertension or hypoglycemia in people, is a commonly used mitoK_ATP channel opener (55). It has the additional effect of inhibiting SDH especially at high doses (15,16,93). Diazoxide readily crosses the blood brain barrier (BBB) and thus is effective in the brain when given intravenously (103). It is not known whether BMS-191095 crosses the BBB, but it is effective in preconditioning the brain when introduced into the cerebral spinal fluid.

Applications of diazoxide or BMS-191095 depolarize mitochondria. However, diazoxide, but not BMS-191095, also causes the liberation of ROS (15,16,55,93), which may be secondary to SDH inhibition. This view is supported by examination of the effects of 3-NPA, a specific inhibitor of SDH, which increases ROS production by mitochondria (15) and changes cerebral vascular tone (56). Nonetheless, the primary actions of diazoxide on the cells of the neurovascular unit are still specific to mitochondria (12, 88), and the associated ROS increase appears to enhance the degree of depolarization (92,94). In contrast, BMS-191095 is very selective for mitoK_ATP channels, does not increase mitochondrial or cellular levels of ROS, and has no known nonspecific effects to complicate interpretation of the results (12,64,86,88).

A potential role for mitochondrial calcium activated potassium (mitoK_Ca) channels in depolarizing mitochondria has been suggested based primarily on the use of the multiple target drug NS1619 (7,35,96). We were the first laboratory to report that NS1619 is vasoactive in cerebral arteries and similar findings come from peripheral arteries (5, 70). Although NS1619 results in mitochondrial depolarization, it seems probable that, at least in neurons, mitochondrial effects are due to initiating factors such as inhibition of Complex I and subsequent increased release of ROS (38,61,62,90). Given the multiple potential sites of action of NS1619 within various cell types, including plasmaK_Ca channels, it is also possible that mitochondrial effects to this drug are secondary to nonmitochondrial events. Nonetheless, additional research in this area is warranted and the development of specific agonists would aid these efforts.

Inhibition of mitoK_ATP channels during disease states might induce or unmask a role for mitoK_Ca channels as a compensatory mechanism. We have shown previously that in cerebral arteries, K_Ca channels are more resilient against the negative effects of chronically or acutely elevated ROS than are K_ATP channels (5).

Role of Mitochondria in Cellular Protection

Preconditioning represents the condition in which transient exposure of cells to an initiating event leads to protection against subsequent, potentially lethal stimuli by limiting ROS availability, suppressing large increases in cytosolic levels of free calcium, and/or by other mechanisms, which promote cell survivability. There are two general types of preconditioning: immediate and delayed. Immediate preconditioning occurs within minutes of the initiating stimulus and lasts for several hours before disappearing, whereas delayed preconditioning takes several hours to develop and persists for several days (61,62,64,91,93). These temporal aspects support the concept that immediate preconditioning primarily involves changes in the activity or function of enzymes, second messengers, and of ion channels already present whereas delayed preconditioning is primarily due to de novo protein synthesis such as increased catalase levels (14, 64). Many subsequent signaling events, including PI3-kinase/Akt activation and NO production, not only induce preconditioning but also promote changes in vascular tone. Postconditioning, in which the initiating event occurs following the onset of the potentially lethal event (130,150), has also been reported and the mechanisms appear to be similar to those that promote preconditioning.

Beginning with our original observation in the neurovascular unit (43), a number of independent studies by other laboratories as well as our own have firmly established that mitochondrial-centered mechanisms are important initiators of the pre- and postconditioning response in neurons, astroglia, and cerebral endothelial cells (40, 42, 50, 63, 66, 70, 79, 83, 92, 97, 104, 111, 124, 127, 130). Mitochondrial-centered preconditioning also occurs in tissues such as myocardium (87), skeletal muscle (69), and peripheral endothelium (9, 105). Important mitochondrial specific targets for inducing pre- and postconditioning by pharmacological approaches include: (i) mitoK_ATP channels located on the inner mitochondrial membrane, (ii) respiratory chain enzymes, and (iii) metabolic substrate limitation.

Although the mitochondrial-initiated mechanisms are not fully understood, production of NO, the activation of protein kinases, transient but modest calcium fluxes, and/or production of ROS appear to be essential signaling events leading to preconditioning (Fig. 10). In a recent study, we found that preconditioning with diazoxide protected cultured neurons against oxygen-glucose deprivation (OGD) via steps beginning with mitochondrial activation and involving production and generation of NO (50, 89). We had previously shown that NO is also a major component in the induction of preconditioning in the intact brain following cortical spreading depression (75) and similar results have been reported by other laboratories (142).

Figure 10.

Schematic illustration showing the primary signaling pathway involving NO production and S6K phosphorylation linking mitochondrial activation with preconditioning of cultured neurons against OGD. (A) All neurons were exposed to OGD. Diazoxide (DZ) protected neurons against OGD. Protection was eliminated by NOS inhibition and restored with NO donors. DZ, diazoxide; SNP, sodium nitroprusside; DEANO, (CH₃CH₂)₂N-N(N=O)O^– Na⁺ × H₂O. Values are mean ± SEM. *P < 0.05, compared to Dz treated, #P < 0.05, compared with untreated or DZ+L-NAME treated neurons. Data adapted from (50). (B) Preconditioning by neurons was eliminated by coadministration of siRNA against S6K. Values are mean ± SEM, *P < 0.05, compared with DZ preconditioning against OGD. Data adapted from (50). (C-E) Preconditioning by diazoxide enhanced the levels of phosphorylated/total of Akt, mTOR, and S6K and coadministration of L-NAME reversed this effect during OGD. Values are mean ± SD. DZ, diazoxide, #P < 0.05, compared with no treatment prior to OGD. Data adapted from (89).

In cultures of primary rat brain microvascular endothelium, we found that postconditioning was as effective as preconditioning against OGD, and that activation of the PI3 kinase/Akt signaling pathway was essential in both types of cellular protection (19). Together with our more recent in vivo data showing endothelial resilience against transient ischemia (14,132), the ability of mitochondria from cerebral vascular endothelium to function at the end of OGD (where almost 50% of cells would eventually die without treatment) and respond to agents, such as BMS-191095, indicates that endothelial mitochondria are a potential target in stroke patients.

Reductions in key metabolic substrates such as glucose and oxygen can induce preconditioning (3,63,115) as well as changes in cerebral vascular tone, but downstream signaling mechanisms are not clearly understood. Transient withdrawal of glucose, the major energy substrate for neurons, as well as removal of amino acids from the culture medium results in mitochondrial depolarization, reduced ATP production, and the development of delayed tolerance against various insults, such as OGD, glutamate excitotoxicity, and exogenous hydrogen peroxide (63). Breakdown products of ATP: adenosine, AMP, ADP, as well as ATP itself, are vasoactive stimuli in cerebral arteries (36, 123).

Although some authors have presented a case for a role of mitoK_Ca channels in promoting preconditioning based primarily on the use of the multiple target drug NS1619 (25,35), the experimental evidence is not convincing due to the absence of a specific mitoK_Ca channel opener and the numerous nonspecific effects of NS1619. We were the first to show that NS1619 is neuroprotective (61,62) and vasoactive in cerebral arteries (5, 74), and that preconditioning of neurons by this agent was probably due to inhibition of Complex I or other mechanisms rather than a mitoK_Ca channel-specific effect (61, 62, 90, 96). However, mechanisms related to mitoK_Ca channels might be different in the heart (90).

A number of other approaches targeting mitochondria, have been used to protect cells following stress. For example, lipid permeable, positively charged antioxidant agents, which are attracted to the negative membrane potential of the mitochondria, have been shown to protect the brain against anoxic, ischemic, hypertensive, and chemical injury (14,39,67,128,136). In addition, cardiolipin-directed compounds have promise as therapeutic agents to protect and restore mitochondrial bioenergics and morphology (139). With the current focus on targeting mitochondria, especially in cancer biology, it seems likely that additional approaches will be examined in the future.

Mitochondria and Cerebral Vascular Tone

Our studies of preconditioning have indicated that many of the signaling events associated with the induction of mitochondrial-targeted cellular protection are the same that affect cerebrovascular tone. Although it is known that extracellular ATP, from either glycolysis or oxidative phosphorylation, as well as the metabolites adenosine, AMP, and ADP (3,115) can alter cerebrovascular tone via plasmalemmal purinergic receptors (115,123), only a few studies have examined direct nonpurinergic mitochondrial influences on the diameter of cerebral resistance vessels (26, 85, 86, 88, 89, 131, 132, 148, 149). Thus, the study of mitochondrial-initiated events on the cerebral vasculature is a relatively new field. Only a limited number of studies have examined mitochondrial influences on the cerebral vasculature during disease states, but increasing amounts of information are rapidly becoming available in other systemic circulations (94,95,146,147) and VSM (22,23,114). The majority of studies involving mitochondrial influences on the cerebral vasculature have used pharmacological agents directed toward mitochondria. However, studies in mice showed that a deficiency of MnSOD resulted in increased basal levels of superoxide anion and endothelial dysfunction in cerebral arteries especially with exposure to angiotensin II and aging in male rats (29,38,138).

Vascular smooth muscle

Jaggar and colleagues (25, 148) were the first to show that depolarization of mitochondria by diazoxide promoted relaxation of VSM cells in endothelium-denuded cerebral arteries or freshly dissociated VSM via a mechanism involving production and localized effects of ROS. Thus, diazoxide application enhanced the generation of ROS from mitochondria, which sequentially caused the activation of ryanodine-sensitive Ca²⁺ channels on the SR, the localized generation of Ca²⁺ transients called “Ca²⁺ sparks,” and the opening of adjacent large-conductance plasma BK_Ca channels. Calcium sparks by themselves are insufficient to affect global intracellular Ca²⁺ in the absence of BK^ channels. The resulting K+ efflux due to opening of plasmaBK_Ca channels by calcium sparks leads to VSM hyperpolarization, decreased global intracellular Ca²⁺, and VSM relaxation. We have reported similar findings in endothelium denuded cerebral arteries with diazoxide (85,86). The role of mitochondrial ROS in promoting VSM relaxation of cerebral arteries is also shown from experiments using 3-NPA (85).

We recently found that BMS-191095 induced similar signaling events to diazoxide in VSM without the involvement of ROS-mediated influences (86). Thus, BMS-191095 does not increase cytosolic or mitochondrial levels of ROS in VSM and dilation of endothelium-denuded cerebral arteries to BMS-191095 is not affected by ROS scavengers (Fig. 11). However, BMS-191095 via mitochondrial depolarization increases calcium sparks activity in VSM, the opening of adjacent BK_Ca channels on the plasma membrane application, and relaxation of endothelium-denuded (as well as endothelial intact) cerebral arteries. The reasons for these differences in experimental findings using diazoxide and BMS-191095 are unclear, but cumulatively they underscore the importance and diversity of mitochondrial mechanisms in promoting relaxation of VSM. We speculate that there is direct electrophysiological coupling between mitochondria and SR in VSM. The close physical associations underlying direct coupling of mitochondria and SR are clearly seen with electron microscopy in cerebral VSM (Figs. 1–3).

Figure 11.

Demonstration that BMS-191096 is able to depolarize mitochondria in VSM without eliciting an increase in mitochondrial ROS production. (A) Original tracings showing ESR values for vehicle versus BMS-191095 and diazoxide for denuded cerebral arteries. (B) ESR analysis showed diazoxide but not BMS-191095 increased mitochondrial ROS generation in denuded cerebral arteries. (C and D) Original confocal images and summary data showing that diazoxide but not BMS-191095 increased mitoSOX intensity in VSM. (E and F) Original confocal images and summary data illustrating that BMS-191095 depolarized (loss of intensity) VSM. Data (means ± SEM) and for graphs and images are from (86). *P < 0.05, compared with vehicle.

Endothelium

Mitochondrial content in the cerebral vascular endothelium is higher than in endothelium in other peripheral circulations, probably due to the augmented transport requirements of the BBB (120). We investigated the contribution of mitochondrial factors arising within the endothelium on the integrated response of intact cerebral arteries using several approaches (88). First, removal of the endothelium or inhibition of eNOS with L-NAME reduced the vasodilation to diazoxide and BMS-191095, implying that traditional endothelium-derived factors, such as NO contribute to changes in vascular tone (88). Second, fluorescence and electron spin resonance measurements of NO in intact arteries or cultured cerebral microvascular endothelial cells confirmed the production of NO in response to diazoxide and BMS-191095. Third, fluorescence measurements showed that a global increase in free cytosolic calcium was temporally associated with increased NO production with diazoxide or BMS-191095 application. Fourth, BMS-191095 or diazoxide promotes PI3-kinase/Akt induced NOS phosphorylation, an event which has been shown to activate eNOS and enhance NO production. Thus, BMS-191095 and diazoxide application led to mitochondrial depolarization, activation of eNOS by increased cytosolic calcium as well as the phosphorylation of eNOS, enhanced production of NO and its diffusion to VSM, and relaxation of VSM (88; Fig. 12). The increased production and diffusion of NO from endothelium augments the relaxation due to mitochondrial activation in the VSM (Fig. 12). Mitochondrial depolarization also can lead to the production of constrictor prostanoids (85) and probably other vasoactive agents by endothelium, but the NO effects appear to be more important, at least with BMS-191095.

Figure 12.

Schematic illustration showing signaling pathways linking mitochondrial depolarization in different cell types of the neurovascular unit to changes in cerebral vascular tone. The intrinsic VSM response involves mitochondria-SR interactions resulting in enhanced calcium sparks activity. The extrinsic VSM response involves the effects of vasoactive stimuli such as NO on VSM tone via a cGMP-linked mechanism. *, phosphorylated state.

Gutterman and colleagues (146,149) showed that a selective physiological stimulus, such as increased shear stress is able to directly activate mitochondria in coronary endothelial cells via cytoskeletal connections arising from the plasma membrane and possibly connecting with the outer mitochondrial membrane, and can dilate coronary arteries via generation of mitochondrial-derived hydrogen peroxide. Whether a similar endothelium-specific mechanism operates in response to increases in flow or shear stress in cerebral arteries is unclear. Nonetheless, the common feature is that activation of mitochondria by physiological stimuli or pharmacological agents leads to the liberation of substances by the endothelium, which can secondarily affect VSM tone.

An unexplored concept is whether endothelial mitochondria, localized to specialized endothelial-VSM connections which penetrate the internal elastic lamina, are able to generate calcium or other events which affect VSM tone (82). Another unanswered question is whether mitochondrial-mechanisms arising in pericytes are able to affect the function of endothelium or VSM. A commonly observed feature of pericytes is the presence of relatively large mitochondria which are located very close to or adjacent to the ablumenal endothelial membrane (Fig. 4).

Perivascular and Parenchymal Nerves and Astroglia

New findings indicate that mitochondrial influences from perivascular nerves or adjacent parenchymal neurons may have major effects on cerebral vascular responses. Cerebral arteries receive dense innervation from sympathetic, parasympathetic, and nitroxidergic nerves (4, 13, 33, 141). Additionally, the entire cerebral vasculature is in close contact with adjacent parenchymal neurons, perivascular nerves or astroglia (108,127; Figs. 4–6 and 8). BMS-191095 and diazoxide depolarize mitochondria in cultured cortical neurons and astroglia (50,64, 111, 127) and enhance NO production by these cells (88,89; Fig. 12). As in endothelial cells, opening of mitoK_ATP channels increases intracellular calcium levels and phosphorylates neuronal NOS (89). Furthermore, inhibition of nNOS with 7-NI in perivascular nerves inhibits dilation in both intact and denuded cerebral arteries to BMS-191095 (89). Therefore, perivascular nerves, parenchymal neurons, and astroglia can provide mitochondrial-initiated vasoactive signals to VSM for the final determination of integrated changes in cerebral vascular diameter in response to physiological and pathological challenges (Fig. 12). In particular, with respect to localized neuronal activation, mitochondrial activation of parenchymal neurons and/or astroglia and subsequent production of vasoactive agents may provide the mechanistic basis for the coupling of brain metabolism and blood flow.

Sexual Dimorphism in Mitochondrial Mechanisms

Previous studies, based largely upon expression of mitochondrial proteins, have suggested differences in mitochondrial respiration between male and female arteries and in cerebral vascular endothelial cells treated with estrogen (47, 48), but did not actually measure mitochondrial oxygen consumption rate (OCR) or determine sex-dependent mitochondrial effects on vascular tone. Although an extensive analysis is still being conducted, the general appearance and fine structure of mitochondrial in endothelium and VSM are similar in male and female rat cerebral arteries (131). Similar to males, large clusters or fields of mitochondria together with SR are present in VSM while smaller, usually more singular mitochondria are present in endothelium in cerebral arteries from female rats. Although untested, it appears that the similar mitochondrial morphology of female arteries would likewise support mitochondrial interactions with SR in promoting relaxation in VSM. Also similar to male rats, astroglia, pericytes, perivascular nerves, and parenchymal neurons in female rats are heavily invested with mitochondria (Figs. 2–3 and 8).

Figure 2.

Electron microscopy section of an MCA representative of 10- to 12-week-old, female SD rats showing numerous sites of close association of mitochondria in different VSM cells. Several VSM cells with extensive mitochondrial fields are present, and multiple sites of close approximation of adjacent mitochondria are seen. Similar mitochondrial fields and connections in VSM are seen in male arteries. White arrows show examples of close mitochondrial connections. Black arrow shows a stressed mitochondrion. Magnification is 11,000x.

Although the general mitochondrial morphology is similar in male and female rat cerebral arteries, there are major differences in mitochondrial protein mass, respiration, and function (131). For example, we have found that mitochondrial protein mass, from nuclear as well as mitochondrial DNA coded proteins, is greater in female compared with male rat cerebral arteries. These include representative proteins from the outer mitochondrial membrane (VDAC) as well as proteins from Complexes I-V (Fig. 13). For studies of mitochondrial respiration, we adapted the Seahorse Bioscience XFe²⁴ Analyzer to allow the determination of mitochondrial OCR in freshly isolated cerebral arteries for the first time (131). In these studies, we found that OCR was greatly enhanced in female rat arteries (Fig. 13). We also found that suppression of mitochondrial OCR by NO was greater in male compared with female arteries (131). Finally, we also found that dilator responses to diazoxide were greater in female rat arteries (Fig. 13). The combined evidence from many different perspectives and methods shows that there is considerable sex-related diversity in mitochondrial mass, function, and dynamics in rat cerebral arteries. The underlying basis for these differences are not clear, however it is possible that our data indicate an enhanced capability of the female cerebral vasculature to survive and respond during states of high energy demand, such as pregnancy and cyclical changes in metabolically active sex hormone levels accompanying menstruation. Evidence that extensive cerebral vascular responses, such as remodeling occur during pregnancy is consistent with the above hypothesis (80). Whether sex-dependent differences in mitochondrial dynamics affect cerebral vascular function in human cerebral arteries in health and disease has not been examined, but may be an important, future research topic.

Figure 13.

Sex dependent differences in mitochondrial dynamics in large cerebral arteries from young rats. (A) Oxygen consumption was greater in 8- to 10-week-old SD female than male cerebral arteries during treatment with inhibitors of mitochondrial complexes and FCCP. (B) The protein mass of inner mitochondrial membrane proteins such as those composing Complex I were larger in female compared with male cerebral arteries. (C) The protein mass of outer mitochondrial membrane proteins such as those composing VDAC were larger in female compared with male cerebral arteries. (D) Dilation to diazoxide was greater in female compared with male isolated and pressurized MCAs. Data (means ± SEM) are from (131).* P < 0.05, compared with male.

Vascular Mitochondria in Insulin Resistance

IR often precedes the development of type 2 diabetes by many years and is considered a relatively quiet phase despite modest vascular dysfunction and moderate arterial hypertension. We and others have characterized many of the general cardiovascular effects of this disease (19,51–53,85,87,94,112,119) and have shown that tissue and vascular inflammation and increased basal levels of ROS from both mitochondrial and nonmitochondrial sources are responsible for vascular and cardiac dysfunction (19,119).

We were among the first investigators to demonstrate that IR is able to disrupt functioning of plasmaK_ATP channels in cerebral and peripheral arteries (19,51–53) and, therefore, it is not surprising that mitoK_ATP channels could be affected in a similar manner (85,87). Thus, diazoxide-induced preconditioning failed to protect hearts from ischemia/reperfusion in IR Zucker obese rats, whereas diazoxide limited infarct size in hearts from non-IR rats (87). An additional finding from this study was that infarct size was enhanced in the hearts from Zucker obese compared with lean rats, which is similar to what we (112) and others (10,140) have found in brains of IR, obese mice following transient occlusion of the middle cerebral artery (MCA). These findings indicate that normal protective mechanisms initiated at the level of the mitoK_ATP channels may be impaired in many common disease states and thus the brain and other organs are more at risk during ischemic episodes.

Similar to impaired preconditioning, mitochondrial-dependent vasoactive responses in cerebral arteries are impaired in IR rats (85, 86). This dilation attenuation appears to be due to reduced mitochondrial depolarization of VSM as well as reduced ROS generation and calcium sparks generation in response to diazoxide. In addition, decreased NO production and/or bioavailability appears to contribute to diminished relaxation of VSM.

The potential for the uncoupling of the tight relationship between metabolic need and blood flow in the brain due to impairment of mitoK_ATP channel function associated with IR may explain the increased risk and severity of neurological diseases and strokes in patients suffering from the metabolic syndrome. A possible new therapeutic approach would involve the early detection and treatment of IR individuals toward reducing vascular inflammation and mitochondrial dysfunction prior to progression of this chronic disease. We have shown that treating IR rats with statins was effective in reducing cerebral vascular ROS levels and restoring normal plasmaK_ATP channel function as well as vascular responsiveness (53,112).

Our preliminary data from electron microscopy indicate that mitochondria in large and small cerebral arteries appear normal in 10- to 12-week-old, IR obese male rats compared with lean male rats (Figs. 1–6). This finding is consistent with our published results which show that ROS scavengers or PKC inhibitors (51,52) are able to almost immediately restore normal vascular responsiveness in cerebral arteries from IR animals. It is untested whether prolonged IR or type 2 diabetes results in irreversible suppression of mitoK_ATP channel function as well as promoting changes in mitochondrial morphology and numbers in the cerebral vasculature.

Mitochondria and Cerebral Ischemia

Although mitochondrial failure is a major cause of cell death due to ischemia, mitochondria in surviving endothelium may represent a useful target to prevent further cell death and restore cerebral blood flow even after the onset of stroke. In a recent study, we examined the effects of experimental stroke on MCA responses 2 days following reperfusion, and found that dilation to mitoK_ATP channel opening with diazoxide was retained on the side ipsilateral to the stroke (132; Fig. 14). Mitochondrial mass and mitoDNA were maintained or enhanced in middle cerebral arteries from the stroke side. At this time, dilator and constrictor responses to other, nonmitochondrial stimuli were reduced in middle cerebral arteries on the stroke side (Fig. 14). Retention of dilator responses to mitoK_ATP channel opening appeared to be due primarily to enhanced contributions of endothelium (Fig. 15). Additionally, in separate studies, we have found that postconditioning cultured cerebral vascular endothelium increases viability following anoxic stress (14). Thus, not only are endothelial cells responsive to mitochondrial-induced postconditioning after anoxic stress (14), but cerebral vascular endothelium responsiveness to mitochondrial activators is also retained or enhanced (132). Since occlusive strokes occur unpredictably in people, therapeutic targeting of mitochondria after the onset of ischemic stroke, especially after delayed clot resolution, might be a clinically useful approach to prevent further injury to the endothelium and VSM, correct cerebral hypoperfusion, restore normal cerebral vascular responsiveness and the BBB, and restrict further neuronal and glial cell death.

Figure 14.

Demonstration that transient ischemia reduces dilation of MCAs to acetylcholine, bradykinin, and sodium nitroprusside but not to diazoxide in pressurized, isolated MCAs from 8- to 10-week-old, male rats. Surprisingly, dilator responses to acetylcholine, bradykinin, and especially diazoxide were reduced in arteries not directly exposed to cessation of blood flow. *P < 0.05, side ipsilateral (IPSI) to stroke compared with sham control, †p < 0.05, IPSI compared to sham control, ^‡P < 0.05, compared to contralateral side (CONTRA). Data (means ± SEM) adapted from (113).

Figure 15.

Retained dilation of pressurized, isolated MCAs from 8- to 10-week-old, male rats following ischemia is due to mitochondrial activation in endothelium and not VSM. (A) Approximately one-half of all dilation to diazoxide in Sham, control MCAs is due to endothelial factors while the remainder is due to VSM. In contrast, the retained dilation following ischemia in MCAs is due solely to endothelium. Denudation reduced dilation more in Sham than Ipsi arteries. (B) Phosphorylation of eNOS is greater in previously ischemic MCAs, which is consistent with a greater endothelial contribution to the retained dilation. *P < 0.05, compared with Sham. (C) Calcium sparks activity during basal conditions is reduced in previously ischemic MCAs and does not increase substantially in the presence of diazoxide. Unpublished data (means ± SEM) for panel C were generated using previously published methods (86) from MCA of male SD rats 48 h after 90 min of ischemia. IPSI represents artery that was occluded and Sham represents a corresponding MCA that was not ischemic from a Sham operated rat. *P < 0.05, compared with Sham. n = 14 images for Sham and Ipsi arteries. (D) Representative electron microscopic image shows areas of VSM where mitochondrial damage is apparent which accounts for decreased calcium sparks activity. Data (means ± SEM) for A and B are from (132).

An unexpected finding after experimental stroke was that dilations of the MCA to acetylcholine and bradykinin, in addition to diazoxide, on the nonischemic side contralateral to the stroke, were reduced compared with sham-operated control animals (132; Fig. 14). Constrictor effects to serotonin were also reduced on both the ischemic and nonischemic MCAs compared with sham, control animals (132). Almost all previous studies made conclusions based only on comparison of responses from ischemic versus contralateral sides and without a proper sham control side, and therefore, may have missed important information that can be derived from these studies. Based on our studies showing that the mechanisms of vascular dysfunction of previously ischemic arteries as well as arteries distant from the direct ischemic exposure are different following stroke, we propose that separate therapeutic approaches may be needed to reduce vascular and neuronal injury following strokes.

Conclusions, Significance, and Perspective

A review of the literature indicates that mitochondrial mechanisms are important in the development of cellular protection of the neurovascular unit as well as in the control of cerebral vascular tone. First, individual mitochondrial-initiated influences from endothelium, VSM, perivascular nerves, and parenchymal neurons and perhaps other cells types such as astroglia and pericytes contribute to mitochondrial-initiated relaxation effects of VSM into a final, integrated change in cerebral vascular tone (Figs. 12 and 16). Thus, production of vasoactive factors following activation of mitochondria in response to physiological stimuli in one or several of the cells comprising the neurovascular unit may represent the elusive signaling link between cerebral metabolic rate and blood flow as well as matching cerebral vascular tone to physiological status. Second, signaling events promoting cellular protection and changes in cerebral vascular tone are similar (Figs. 10 and 12), thus establishing the importance of mitochondria in many diverse functions beyond just energy production. Third, considerable sex-dependent differences are apparent in mitochondrial dynamics in cerebral arteries and may suggest a greater adaptability of the female cerebral circulation to changes in physiological status and pregnancy. Fourth, mitochondrial-derived mechanisms that induce changes in cerebral vascular tone are disrupted by chronic disease processes, such as IR or diabetes. Thus, direct or indirect targeting of mitochondria in the early stages of the metabolic syndrome may prove to be an effective treatment to reduce morbidity and mortality in these patients and reduce subsequent risk of cardiovascular disease, strokes, and dementias such as Alzheimer’s disease. Fifth, mitochondrial mechanisms promoting vasodilation and endothelial preconditioning are present even following experimental strokes. Thus, targeting mitochondria following an ischemic injury, especially after a delayed resolution of an occlusion, may restrict further neuronal, vascular, and glia cell death.

Figure 16.

Schematic illustration of mitochondrial influences on cell types within the neurovascular unit. All cell types within the neurovascular unit are capable of affecting cerebral vascular tone through their mitochondrial mechanisms. Although the majority of mitochondria in the neurovascular unit can be simultaneously affected by insults such as ischemia or cortical spreading depression, it is more likely that only limited populations of mitochondrial are activated during changes in physiological status. For example, increases in shear stress would only directly activate mitochondria in endothelium through mechanical means as described by Gutterman and colleagues (146, 149). Also, focal activation of neurons would impact directly only on the mitochondria in these neurons and lead to NO production, and an appropriate increase in local blood flow.

Figure 5.

Electron microscopy section of brain parenchyma showing close approximation of microvessel representative of 10- to 12-week-old, male Zucker lean rats showing glial endfoot with prominent mitochondria. Lean Zucker rats are phenotypically normal and the mitochondria of the cerebral vasculature show no obvious differences with non-IR rat strains. Magnification 11,000x.