Cerebrovascular Changes in Substrate Supply
Changes in the cerebrovascular system associated with aging may underlie the increased incidence of ischemic stroke. Structural alteration of the vasculature such as narrowing of the intracranial arteries or small vessel occlusion due to severe atherosclerosis may increase the susceptibility to substrate deprivation. In addition, enhanced vasoconstriction and impaired vasodilatation, in response to increased energy demand, result in a decrease of cerebral blood flow, reduced CMRO2
and CGU even in healthy aging individuals [70
]. After an ischemic event, these alterations of vascular reactivity may limit the energy supply during reperfusion at the time at which tissue energy demand is extremely elevated, leading to more extensive damage [71
]. Interestingly, investigators have found that in animals exposed to brain ischemia the rate of lethality increased with age [39
]; moreover, in brain slices isolated from aged animals, the incidence of cell death and synaptic failure after exposure to hypoxic/ischemic insult is higher compared to slices isolated from young adult animals [26
]. These observations indicate that, in addition to systemic changes, metabolic alterations [74
] at the cellular level of both neurons and astrocytes may also contribute to the increased susceptibility of aged individuals to ischemiareperfusion injury.
Cellular Events Underlying Ischemia
The cascade of cellular events occurring during ischemia includes a dramatic decrease of neuronal ATP, membrane depolarization, glutamate release and increase in intracellular Ca2+
. The ion gradients collapse and within a few minutes all neurons depolarize due to the failure of the Na+
ATPase and other transport systems dependent on ATP [75
]. These events can be reversible following the ischemic episode during reperfusion, or they can trigger neuronal death. Some of the metabolic changes, such as the increase in intracellular Ca2+
, are critical in determining irreversible damage to the neurons after ischemia. The large increase in cytosolic Ca2+
causes the generation of free radicals and the activation of intracellular proteases and kinases that initiate cell death mechanisms ([76
] for review). High intracellular Ca2+
can also trigger mitochondrial injury. During ischemia and reperfusion, excessive mitochondrial Ca2+
sequestration compromises mitochondrial function, resulting in respiratory inhibition, ROS formation, dissipation of the transmembrane mitochondrial gradient and transient loss of mitochondrial membrane integrity (mitochondrial permeability transition (MPT)) [77
] (). This chain of events further results in the efflux of small molecules and metabolites from the mitochondria to the cytosol including: Ca2,
/NADH, and cytochrome c. After reperfusion, depending on the degree of ATP depletion and cytochrome c release, different downstream cell-death pathways can be activated and cells can undergo either apoptosis or necrosis [78
Energy Homeostasis and Neuronal Vulnerability
Although the mechanisms behind the increased vulnerability to ischemia associated with aging are not well understood, investigators have focused their attention especially on mitochondrial dysfunction, a decrease in glucose utilization [27
], increased oxidative stress, deregulation of ionic homeostasis, and compromised glial function. See for a summary of the cellular events that investigators have proposed contributing to increased neuronal vulnerability to hypoxic/ischemic insult with aging.
Despite the age-dependent deficiency in the electron transport chain (as discussed above), ATP production during resting conditions does not appear to be compromised, as several investigators found no significant age related changes in the concentration of high energy substrates under normoxic condition [23
]. For example, measurements conducted using the whole brain or acutely isolated brain slices have found that ATP and PCr levels were not altered in either middle aged (14–19 months old) or aged (24–29 month) rats, compared to younger rats. However, reports have suggested that a drop in ATP levels may occur earlier after exposure to hypoxia in aged subjects, leading to earlier failure of the membrane ion gradient and depolarization. Roberts et al., reported that during anoxia in hippocampal tissue slices isolated from aged rats the latency for developing anoxic depolarization (AD) or hypoxic spreading depression (HSD) was shorter in aged rats compared to younger rats [27
]. In this case, the investigators suggested that aging tissue has a decreased ability to up-regulate anaerobic glycolysis to provide ATP for ion pumping, leading to a faster increase of extracellular K+
during hypoxia. These findings, however, are in conflict with a more recent study that did not find a significant difference in the time to HSD between >22 months old and younger animals [26
]. Consistent with the notion that aging compromises the ability to respond to increased metabolic demand, slices of older rodents show a slower rate of ATP recovery after they are exposed to transient global ischemia [81
], which is accompanied by a slower recovery of the pH level in the tissue.
Early during reoxygenation, mitochondria accelerate the rate of oxidative metabolisms utilizing all the available oxygen to generate ATP needed to restore the ionic homeostasis and the membrane potential. Tissue Po2
measurements demonstrate that in young animals following hypoxia of 2.5 min duration (post HSD) tissue oxygen levels remained severely depressed after reoxygenation due to increased oxygen uptake, and the recovery of tissue Po2
to pre-hypoxic levels is slow [82
]. However, in 22-month old rats exposed to the same insult, the tissue Po2
recovered earlier after reoxygenation, reaching levels that were significantly higher than the pre-hypoxic baseline levels [26
]. These observations suggest that in aged rats, tissue oxygen uptake (and mitochondrial oxygen utilization) was decreased probably due to the inability of the mitochondria to fully restore respiratory function after reoxygenation in spite of the immediate availability of an excess of oxygen.
In young rats, mitochondrial respiratory capacity is impaired during ischemia and recovers toward control levels within the first hours after reperfusion. Oxygen uptake measurements performed in brain homogenates collected from the focal and perifocal tissue at different time points after exposure to middle cerebral artery occlusion (in young rats), showed a progressive decline of ADP stimulated and uncoupled respiration; in contrast, basal respiration was preserved. Similar experiments also showed that mitochondria develop lower membrane potential during ischemia and early reperfusion. The extent of mitochondrial recovery during reperfusion depends on the severity of the insult; in mitochondria collected from the core of the ischemic lesion, the recovery is slower and sometimes incomplete [83
]. Despite a transient recovery, later during reperfusion mitochondria undergo a secondary deterioration of the respiratory function which precedes and contributes to the development of irreversible neuronal injury. For example, in vulnerable brain regions, such as the area CA1 in the hippocampus, mitochondrial respiratory capacity declines later after exposure to transient global ischemia, leading to delayed cell death [84
]. Because of underlying dysfunction, exposure to hypoxia/ischemia may have more detrimental effects in mitochondria of aged rats, resulting in a more severe impairment of the respiratory function. If mitochondrial dysfunction persists during reperfusion, the rate of ATP production may fall below ATP demand resulting in metabolic failure.
Foster et al. [26
] showed that synaptic responses in hippocampal slices isolated from aging rats were more vulnerable to hypoxia compared to younger rats; for example, after hippocampal slices were exposed to hypoxia and reoxygenated at 2.5 min post-HSD occurrence, the fractional recovery of orthodromic field potentials (fEPSPs) was significantly lower in slices from 22-month old rats compared to younger rats (). Note that the time to onset of HSD was similar in young and aged rats, but slices from aged rats were much more sensitive to prolonged periods of post-HSD hypoxia (ie, > 30 sec) indicating much less reserve metabolic function in the aged tissue slices in the persistent absence of oxygen.
Fig.3: The vulnerability of synaptic responses to hypoxia increases with age. Hippocampal slices from 1–2, 3–6, 12–20, >22 months old rats were exposed to varying lengths of hypoxia following the induction of hypoxic spreading (more ...)
During reoxygenation, as the tissue Po2
recovers there is a progressive switch of the NADH/NAD+
redox state toward oxidation due to elevated respiration, and eventually NADH fluorescence falls below baseline levels, indicating hyperoxidation and mitochondrial damage. Because the decrease of NADH fluorescence is partially irreversible, several investigators have proposed that a net loss of NAD+
from the mitochondrial pool due to induction of MPT may contribute to the hyperoxidation [26
]. Aged rats were more vulnerable to hyperoxidation after hypoxia compared to young rats exposed to similar insult [26
] consistent with the hypothesis that the sensitivity to permeability transition pore (PTP) opening increases with age [86
]. Several factors that contribute to MPT and hyperoxidation may be exacerbated by aging, in particular increased ROS production, altered calcium buffer ability and mitochondrial dysfunction.
As described earlier, calcium accumulation during ischemia and ROS bursts occurring during reperfusion appears to facilitate the opening of mitochondria PTP, which facilitates the diffusion of NAD+
from the mitochondria to the cytosol. This event is usually accompanied by a transient collapse of the mitochondrial membrane potential and proton gradient, which result in the uncoupling of the oxidative phosphorylation and interruption of ATP synthesis. Despite reperfusion and recovery of the mitochondrial membrane potential, the induction of MPT initiates a cascade of events that contribute to irreversible neuronal injury. Consistent with the hypothesis that these events may be exacerbated by aging, investigators have reported that cellular markers of degeneration were higher in hippocampal slices from 24-month old rats, which release higher levels of LDH after exposure to analogous OGD insults compared to middle aged or young adult animals [87
Recently, investigators have demonstrated that induction of MPT exacerbated ROS and NO generation after ischemia [88
], which are both key mediators of apoptotic cell death. Moreover, oxidative stress after ischemia contributes to further cytosolic/nuclear NAD loss via the activation of poly-ADP ribose polymerase-1(PARP-1); this enzyme hydrolyses its substrate nicotinamide adenine dinucleotide (NAD+
) to nicotinamide and transfers poly (ADP-ribose) chains to a variety of other nuclear proteins to repair ROS-induced DNA damage during reperfusion [26
]. Availability of metabolic cofactors, such as NAD+
, is critical during reperfusion, when the mitochondria accelerate the rate of oxidative metabolism to rapidly restore ATP levels and to maintain cellular function. Depletion of NAD+
causes inhibition of several energy pathways including glycolysis, TCA cycle, and oxidative phosphorylation. Rats treated with nicotinamide after stroke had elevated NAD+
levels in the brain several hours after the injury [89
] and attenuated neuronal death.
Limiting the loss of ATP levels is very important for neuronal function when energy production is impaired; for example, during hypoxia, which results in a rapid loss ionic homeostasis [90
]. Maintenance of the electrochemical gradient across the membrane largely depends on the work of the NA+
ATP-ase and is critical for brain function, such as generating, processing and transmitting impulses. Investigators have reported that the activity of the ATP-dependent membrane pump is enhanced after ischemia both in vivo
] and in vitro
], and the activation persists for several hours to allow for the recovery of transmembrane ion gradient. In aging rats, investigators reported a deficit in the up-regulation of the activity of the NA+
ATP-ase 1hr after ischemia compared to younger rats, possibly due to insufficient levels of ATP. Failure to restore ionic homeostasis early after an ischemic/hypoxic insult delays the recovery of synaptic transmission, exacerbates the accumulation of intracellular Ca2+
and can contribute to the activation of a downstream cell death signal [91
] such as the release of cytochrome c from the mitochondria.
Interventions during energy deprivation that limit ATP depletion or promote ATP recovery can also result in long-term protection by preventing the release of cytochrome-c and further mitochondrial dysfunction that may take place during reoxygenation [92
], probably by restoring earlier ionic homeostasis and limiting intracellular and intra-mitochondrial calcium overload [92
]. Especially in an aging model, restricting Ca2+
uptake during hypoxia prevented hyperoxidation and improved the recovery of synaptic transmission in hippocampal slices of 22-month old rat exposed to hypoxia and reoxygenated 2.5 min post HSD [26