In this work, we show that sepsis influences both cerebral and systemic responses to NCSz. The enhanced sepsis-induced EEG power during seizure is accompanied by a reduced NVC response.
The microcirculatory changes induced by sepsis, such as the reduction in the proportion of cortical perfused vessels 10, the astrocyte end-feet 11 and pericytes 12 detachment from the vessel walls and increased the blood-brain-barrier permeability 13 could alter the extracellular milieu of the brain, interfering with the neurovascular unit. Moreover, release of cytokines with a high vasoconstriction power, such as endothelin-1 14, might move the physiological balance towards arteriolar vasoconstriction, either directly or by a reduction in endothelial nitric oxide production 15. Consistent with these hypotheses, we showed that the seizure-induced cerebral vascular response in the sepsis group is relatively flattened. In fact, despite a more important neuronal and vascular activation, the Eγ/CBFv ratio was greater in the sepsis group, suggesting that the increase in neuronal activity was associated with a proportionally smaller CBFv variation than in shams. Possibly because of this reduced vascular response to neuronal activation, the rise in the PbtO2 tended to be delayed. These findings agree with previous studies that suggested that sepsis alters but not completely abolishes the NVC 16,17. Indeed, we showed a persistent positive correlation between neuronal activation and vascular response in both groups. As sepsis progresses, more severe impairment of NVC, as previously demonstrated18 could be partially responsible for the alteration of brain function, which might prevent seizure occurrence, as observed in this study.
Interestingly, the altered NVC does not translate into an immediate neuronal dysfunction or substantial tissue hypoxia. Auxiliary sources of energy than mitochondrial oxidative respiration, such lactate or ketone bodies, might thus support neuronal activity 19,20 in the context of reduced O2 availability. In line with this hypothesis, the physiological PbtO2 dip after seizure offset 21 was relatively smaller in sepsis animals, suggesting a reduced O2 consumption. Since we did not found a reduction in cortical activity in sepsis animals (Table 1), we deemed less likely that the smaller changes in the PbtO2 would be due to a reduced cortical metabolic demand, as it has been shown to occur in sepsis22. As sepsis progresses, this cerebrovascular inefficiency could be partially responsible for the alteration of brain function, which might prevent seizure occurrence.
Animal studies have shown that systemic inflammation enhances cerebral excitability, as revealed by an increase in seizure susceptibility 23. Innate immune cells (i.e. microglia) could play a major role in this pathological response 24 and previous studies have demonstrated microglial activation in sepsis 25–27. Moreover, some human studies suggested that sepsis was a significant risk factor for NCSz 1 and probably for NCSE 2. In line with them, an increase of seizure susceptibility would have been expected in the sepsis groups; instead, although a prominent ictal Eγ power in sepsis animals, no seizures were observed in the septic shock group, despite a similar penicillin dose. In severe septic animals, brain function and perfusion could be altered to such an extent to reduce the ability of neurons to fire synchronously and thus prevent seizure occurrence, as suggested in a human study 28. Using the fast/delta frequency ratio as a surrogate for cortical neuronal activity 29, we showed that septic shock animals presented a lower ratio than sham or sepsis group, suggesting an impairment of neuronal function. Brain dysfunction is a well-known complication of sepsis (i.e. sepsis-associated encephalopathy - SAE) 30 and it has been shown that increasing severity of brain dysfunction was associated with progressing slowing of brain activity 31, resulting in a decrease in the fast/delta ratio. The presence of a sepsis-induced NVC alteration, as we have shown with the reduced hemodynamic response to the increase in Eγ power in sepsis animals, might play a crucial role in SAE development. In fact, as the fast/delta ratio was not statistically different between sham and sepsis animals, our data support the hypothesis that, in sepsis, NVC impairment precedes neuronal dysfunction 6, which successively develops when septic shock occurs 18. Since no seizure occurred in septic shock animals, we were not able to test the NVC in this scenario and in particular to answer the question of whether seizures are associated with a reduction in tissue oxygenation during septic shock.
We used the NMM to estimate if a difference in temporal evolution of the synaptic activity of the modeled neuronal populations might explain the differences in neuronal activation and in the cerebrovascular hemodynamic response in septic animals. It has been shown that sepsis induces variations in important neurotransmitter metabolisms, such as those of acetylcholine 32, noradrenaline 33 and gamma-aminobutyric acid (GABA) 34, and this might play a role in SAE. For all the analyzed parameters, we noticed a deflection during seizure. These alterations are consistent with what was previously reported in the literature 35 and they suggest an excitation/inhibition imbalance leading to seizure. Of note, the variations were delayed and blunted in the sepsis group, suggesting a certain extent of synaptic impairment induced by sepsis which might not be homogenous between neuronal populations as it happens for ischemic insults36. Therefore, the activity of the fast somatic-projecting inhibitory interneurons, which are described by the fast inhibitory synaptic gain G in the NMM model, was less reduced in the sepsis group. Since these interneurons play a crucial role in inducing gamma oscillations 37, this finding might partially explain the more prominent Eγ power in sepsis. Furthermore, we may speculate that the delayed activation of neural populations in sepsis might explain the delayed seizure-induced peak in CBFv increase we recorded. Finally, these excitation/inhibition imbalance might furtherly worst as sepsis progress, contributing to the negative influence of an altered NVC in preventing seizure occurrence in septic shock.
The ictal systemic hemodynamic response differences between groups are intriguing but hard to explain. Although cardiovascular effects of NCSz are a matter of debate, sympathetic outflow seems to be enhanced during seizures 38. The systemic cardiovascular response in the sham group reflects the physiologic effect of sympathetic stimulation leading to a slight increase in MAP accompanied by a chrono- and inotropic cardiac effect39. In the sepsis group, an increased α adrenergic receptor (AR) activity40 might mediate a rise in MAP by systemic vasoconstriction, inducing a baroreceptor reflex activation and, thus, a reduction in HR and COEST. Moreover, an adrenal insufficiency, which has been reported in sepsis41, may reduce the positive cardiac adrenergic effect of adrenaline; the rise in BP would no longer be combined with a positive chrono- et inotropic effect but by a negative one mediated by baroreceptor reflex. Data interpretation might be furtherly complicated by the interaction of other mechanisms influencing cardiovascular physiology, such as arginine-vasopressin (AVP) and the renin-angiotensin systems (RAS). It has been shown that they share superimposable inotropic and peripheral-vasoconstriction effects in healthy subjects 42,43. Therefore, despite an increase in AVP has been reported after convulsive seizures44, it is challenging to establish if the rise in plasma concentration is a direct effect of seizure or it is an epiphenomena of the physiologic anomalies accompanying convulsion (i.e. hypotension, hypoxia, autonomic nervous system activation 45). Moreover, the role of these hormones in non-convulsive seizure is unknown. Several studies has shown that sepsis induces a depletion of plasmatic levels of AVP and a downregulation of angiotensin receptors in vascular bed46,47. It is thus conceivable that the sepsis-induced alteration in these physiologic systems might influence the cardiovascular response to seizure that we recorded in sepsis animals. Further research is needed to explore the ictal variations of AVP and RAS in non-convulsive seizures.
Previous studies have essentially focused on local cerebral mechanisms of CBF regulation, especially NVC, to describe the vascular response to seizure48,49. However, evidence from both humans and animals experiments suggests a close relationship between CO and CBF50 and a study in septic patients showed a linear relationship between cardiac index and cerebral blood flow51. Despite the difficulty to distinguish the net effects of cerebral and systemic factors influencing CBF, we may speculate that the alteration in systemic hemodynamic recorded in sepsis animal, especially the reduction in the ictal COEST, might negatively influence the physiologic CBF response to neuronal activation.
Our study presents several limitations. First, regarding the latency to seizure onset in septic shock group, one could speculate that, since a neuronal dysfunction occurs in severe sepsis, the time necessary to induce seizure in the septic shock group could have taken longer than in other groups. Since specific sepsis therapies, such as antibiotics, might enhance seizure excitability52, we preferred not to employ them; thus, this hypothesis could not be verified since septic shock animals spontaneously succumbed. However, human data seem to support our presumption that severe sepsis-induced cerebral dysfunction prevents seizure occurrence28. Secondly, the variations in tissue oxygenation we recorded are smaller than those reported in other animal studies48,49. Our data might be the average of the ictal focus and surrounding tissue PbtO2 variations, which are not identical and, in fact, often opposite. Furthermore, since we did not measure any metabolic parameters, we could not definitely conclude to a reduced oxygenation consumption. Of note, no specific intra-group variations or inter-groups differences in PbtO2 values were recorded during resting-state conditions. Thirdly, NMM privileges the description of key mechanisms of neuronal networks using simplified assumptions and empirical priors53, which has been successful in reproducing EEG signals in patients54. Thus, it is possible that the set of parameters fitting humans may not be appropriate for our experimental model. Finally, the influence of cerebral autoregulation (CA), defined as the capacity of the brain to maintain an adequate cerebral blood flow despite variations in cerebral perfusion pressure55, could be considered as a confounding factor in the interpretation of the link between systemic and local variables. As sepsis and seizures seem to alter CA56,57, CBFv variations could be influenced by MAP fluctuations rather than by the sepsis-induced NVC alteration. Moreover, since we did not find significant differences in the correlation between EEG/CBFv ratios and ictal-induced COEST variations between groups, the negative influence of the reduction in the ictal COEST on the CBF response to neuronal activation remains a matter of speculation.