In contrast to the conventional injection model, we developed a model that can control the severity of SAH by controlling ICP. The results of Experiment 1 showed that cortical depolarization occurred in 88.9% of cases, and a duration of depolarization of 20 min or more occurred in 44.4% of cases. The duration of depolarization was significantly correlated with peak extracellular glutamate levels. The extracellular glutamate levels were also significantly correlated with worsening neurological scores and neuronal injury. The results of Experiment 2 showed that cortical depolarization occurred in 100% of cases, but a longer than 20 min duration of depolarization was observed in only 28.6% of cases despite the comparable ICP. CPP recovered earlier in the HT group. The extracellular glutamate levels had a lower prevalence than those in the NT group.
Early brain injury and physiological parameters
Early brain injury occurs as a result of pathophysiological mechanisms triggered by the combination of mechanical trauma and ischemic injury during the first 72 h after SAH which worsens the outcome. First, when an aneurysm ruptures, the arachnoid space is filled with hematoma and the ICP increases to over 150 mmHg12. The compression from the hematoma filling the subarachnoid space causes acute arterial constriction. This, in turn, causes a marked CBF decrease13. Subsequently, changes including brain edema, inflammation, oxidative stress, microvascular embolism, CSD, and apoptosis cause EBI4,14,15. Our blood injection model using ICP control successfully showed dynamic changes in the membrane potentials, ICP, MAP, CPP, CBF, and extracellular glutamate levels within 1 h after SAH, and these parameters are strongly associated with neuronal injuries.
In a previous study, the duration of depolarization was significantly correlated with the severity of neurological injury10. The results of Experiment 1 also indicated that the duration of depolarization has a strong impact on the aggravation of the neuronal injury. In a retrospective study on human SAH, severe ICP elevation was the primary cause of early and prolonged loss of consciousness at the onset of SAH, and loss of consciousness was an important predictor of poor neurological outcome16. Loss of consciousness is caused by electroencephalographic suppression due to cortical depolarization, supporting the fact that the longer the duration of depolarization, the worse the neurological outcome.
Acute vasoconstriction and cerebral autoregulation
Under normal autoregulation, elevation of MAP induces vasoconstriction and reduces the cerebrovascular beds. Next, CBF is maintained in the normal range. In the case of a severely damaged brain in which cerebrovascular autoregulation is disrupted, CBF increases in proportion to the ABP rise by passive dilatation. This leads to brain edema and ICP increase, which results in CPP decrease17. In the hyperacute stage of SAH, acute vasoconstriction occurs independently of the changes in ICP and CPP, and is associated with CBF decrease and persistent elevation of extracellular glutamate18,19,20. The subsequent disruption of cerebrovascular autoregulation contributes to EBI14. Sukhotinsky et al. modulated CPP by controlling MAP, and examined the influence of CPP on the duration of CSD by KCl application. They showed that induced hypertension significantly shortened the CSD recovery, independent of tissue oxygenation, by facilitating glucose delivery and extracellular K clearance. They concluded that CPP is a critical determinant of CSD duration in the compromised brain, as seen in cases of ischemic stroke and SAH21.
The results of Experiment 1 showed that CPP and CBF at 10 min after SAH were significantly correlated with peak extracellular glutamate levels, and extracellular glutamate levels plateaued just 20 min after SAH. This indicates that the impairment of cerebrovascular autoregulation after acute vasoconstriction and the subsequent CBF decrease have a big impact on the prolonged duration of depolarization and neuronal damage. It appears that the fate of SAH patients may be decided within 30 min as a result of ultra-early brain injury.
Extracellular glutamate levels and therapeutic time window
Under normal conditions, glutamate can be synthesized from glutamine or α-ketoglutarate. After glutamate is released into the synapse, it is removed by excitatory amino acid transporters (EAATs) on the pre- and postsynaptic membranes and also glial cells. EAAT3 is located on the postsynaptic membrane, and EAAT1 and EAAT2 are located on glial cell membranes. Under ischemic conditions, disruptions to Na+, K+, and pH gradients will cause transporters to function in reverse, leading to elevated extracellular glutamate concentrations22,23. The increase in extracellular glutamate levels plays an important role in neuronal injury because the excessive release of extracellular glutamate causes an overload of intracellular Ca2+ via post-synaptic N-methyl-D-aspartate (NMDA) receptors. An increase in Ca2+ concentration activates Ca2+-dependent enzymes, resulting in neuronal injury.
In an experimental severe-ischemic model, extracellular glutamate levels started to rise 4 min after depolarization, and reached maximum levels 12 min after depolarization24. In our injection model, we conducted detailed measurements every 2 min. We first showed that the increase in extracellular glutamate levels started 10 min after injection (i.e., approximately 8 min after depolarization), indicating a slower rise in extracellular glutamate levels compared to ischemic injury. Because CBF recovered to 30 to 40% of the baseline after initial mechanical insult and acute vasoconstriction in the SAH model, the elevation of glutamate concentration in the extracellular space might be delayed compared to that in the cerebral ischemic model. In addition, multifactorial mechanisms, such as trapped hematoma, brain swelling, or ICP increase, may also be associated with delayed glutamate release.
Our results indicate that the therapeutic time window in SAH may be longer than that in cerebral ischemia. Fifty percent of neuronal cells were injured around 20 min after injection; therefore, in the experimental model, 20 min might be a therapeutic target that we can aim for to prevent the elevation of extracellular glutamate and neuronal injury.
Brain hypothermia targeting EBI
The neuroprotective effect of brain hypothermia for SAH has been reported in several studies25,26. Recent animal studies have shown that mild hypothermia has the potential to inhibit secondary brain injury by reversing CPP-independent hypoperfusion, preventing brain edema, and reducing vasospasm. Shubert et al. studied the effect of hypothermia using microdialysis in an SAH rat model. They reported that hypothermia ameliorated the acute hypoperfusion and dysfunction of cerebral autoregulation and suppressed the release of excitatory amino acids, such as glutamate, aspartate, and histidine27. In another study using a traumatic brain injury model, hypothermia was shown to improve cerebral autoregulation and preserve vascular dilatation in response to hypoperfusion28.
Our results of Experiment 2 showed that brain hypothermia within 20 minutes contributed to the shorter duration of depolarization, earlier recovery of CPP, and reduced elevation of extracellular glutamate concentrations compared to normothermia, despite comparable peak ICP values. Brain hypothermia may inhibit glutamate release in the extracellular space and activate the Na+/K+/ATP pump by suppressing energy metabolism, inducing early repolarization and preventing brain edema due to the influx of Na+ and Ca2+. In addition, hypothermia may ameliorate MAP and CPP by suppressing acute vasoconstriction and the subsequent cerebral autoregulation, which may, in turn, facilitate glucose delivery and restore the ionic gradients.
Probit analysis showed that the duration of depolarization that caused 50% of neuronal injury is 22.4 min in a perforation model10, and 16.5 min in an injection model. This indicates that the early introduction of neuroprotective therapy such as brain hypothermia within 20 min after SAH inhibits the excessive release of extracellular glutamate and neuronal injuries. Because this study was conducted using rats, the cutoff value of the time window for EBI-targeting therapy in humans remains unknown and further research is needed.
There were several limitations in this study. First, because the observation period was limited to 60 min after SAH, any changes that took place after 60 min were not evaluated. ICP tended to increase during the 60-min period, and it is possible that some cases may have developed brain swelling and herniation after observation, resulting in cerebral ischemia and second depolarization. In addition, we did not investigate whether duration of depolarization and elevation of extracellular glutamate levels were associated with chronic neurological deficits and long-term mortality because we sacrificed the rats 24 hours after SAH. It is necessary to investigate the relationship with long-term outcome in the future. Second, after 60 min of observation, most cases had brain herniation from the burr hole due to increased ICP, which may have caused cerebral contusion at the bony margin of the burr hole and local glutamate elevation. To minimize the effect of brain herniation, a DC electrode and microdialysis probe were inserted into the same burr hole. Moreover, it is possible that the neuronal damage was caused by the insertion of the electrode or microdialysis probe itself, and we should have established a control group in which only the electrode and microdialysis probe were inserted without SAH. In order to minimize brain damage during probe insertion, we inserted a microdialysis probe gently after incision of pia mater. The pilot study showed that the elevation of extracellular glutamate levels due to probe insertion disappeared within 30 minutes. Thus, the influence of mechanical injury is not considered to be a concern. Third, in this study, body temperature was managed with rectal and epidural temperatures to avoid brain injury caused by inserting a temperature sensor into the brain cortex, but there was some discrepancy between epidural temperature and brain temperature. In our pilot study, epidural temperature remained under 30℃ during the 1-h observation period, but brain temperature began to increase after pharyngeal cooling was discontinued because of blood flow. Fourth, due to our study design, Experiments 1 and 2 were performed consecutively and not randomized. Last, in this study, we started brain hypothermia immediately after depolarization, but this is not appropriate for actual clinical situations. To determine the optimum therapeutic time window, we plan to examine when to start hypothermia therapy to prevent glutamate release and neuronal injury.