Preconditioning paradigms and pathways in the brain

MOLECULAR PRECONDITIONING PATHWAYS

Mechanistically, cellular preconditioning can be subdivided into intrinsic neuronal pathways (preventing excitotoxic damage, signaling through anti-apoptotic molecules, and treatment by neurotrophic factors) or extrinsic nonneuronal pathways (peripheral cytokine production, microglial activation, and regulation of the cerebrovascular system). Several neuroprotective molecules are expressed and signal through multiple cell types both within and peripheral to the brain, so that assigning an exact source and paradigm for preconditioning pathways has proven difficult.

NMDA receptor activation and excitotoxicity protection

In neurons, ischemic tolerance is mediated largely by the activation of the N-methyl-d-aspartate (NMDA) glutamate receptors through increases in intracellular calcium.^50–52 Although glutamate receptor activation is generally believed to be responsible for much of the neuronal damage caused by excitotoxicity, it appears to also be implicated in the establishment of preconditioning. One study demonstrated that exposure of cortical cell cultures to low levels of glutamate activated NMDA receptors in preconditioning.⁵⁰ In addition, preconditioning by oxygen-glucose deprivation was blocked when an NMDA antagonist was applied. NMDA receptor activation can induce a tolerant state through rapid adaptation of the voltage-dependent calcium flux. In addition, activation of NMDA receptors leads to rapid release of brain-derived neurotrophic factor, which then binds to and activates its cognate receptor, receptor tyrosine kinase B. Both NMDA and tyrosine kinase B receptors activate nuclear factor–kappa B (NFκB), a transcription factor involved in protecting neurons against insults. In sublethal ischemic preconditioning, activation of NFκB and its translocation from the cytosol to the nucleus was required for the development of late cerebral protection against severe ischemia or epilepsy.⁵³ Other key mediators involved in synaptic NMDA receptor–dependent neuroprotection are phosphatidylinositol 3-kinase (PI3K), Akt, and glycogen synthase kinase 3-beta.⁵⁴

Preconditioning with cortical spreading depression results in the downregulation of the excitatory amino acid transporters EAAT1 and EAAT2 from cerebral cortex plasma membranes.⁵⁵ Although these transporters are normally involved in glutamate uptake, it has been suggested that the influx of sodium that occurs during excitotoxicity may cause their reversal and result in additional glutamate release. Downregulating these transporters may thus contribute to ischemic tolerance.

Nitric oxide

Nitric oxide (NO) may play a key role as a mediator of the neuronal ischemic preconditioning response, either in conjunction with or independent of NMDA receptor activation. Both the inhibition of nitric oxide synthase (NOS) and the scavenging of NO during preconditioning significantly attenuated the induced neuronal tolerance, and neither endothelial NOS nor neuronal NOS knockout mice showed protection from rapid ischemic preconditioning.^56,57 Treatment with the inducible NOS (iNOS) inhibitor aminoguanidine abolished the induced protection. The mechanisms responsible for NO-induced tolerance are not clear. Downregulation of the glutamate transporter GLT-1 might play a role.⁵⁸ A common link to NMDA receptor activation and NO is p21^ras (Ras). Preconditioning induces p21^ras activation in an NMDA- and NO-dependent manner and leads to the downstream activation of Raf kinase, mitogen-activated protein kinases, and extracellular regulated kinase.⁵⁹ Inhibition of these kinases attenuates subsequent protection from ischemia.^60,61 Pharmacologic inhibition of Ras, as well as a dominant negative Ras mutant, blocked preconditioning, whereas a constitutively active form of Ras promoted neuroprotection against lethal insults. An important consideration regarding NO is also that preconditioning by volatile anesthetics appears to involve NO pathways.⁹

NO and reactive oxygen species (ROS) are also implicated in regulating the peripheral cerebrovascular system. Ischemia generated by occlusion of the middle cerebral artery causes defects in cerebrovascular function for not only the infarcted area but also the surrounding ischemic region. LPS preconditioning has been reported in some cases to increase this regional cerebral blood flow both before and after ischemia.^{1,21,36,62–64} LPS also improves microvascular perfusion.^33,64 It was recently reported that LPS-stimulated cerebral blood flow is induced through reactive oxygen and nitrogen species (ROS or NO).¹ Mouse knockouts of iNOS (NO production) or of the nox2 subunit of NADPH oxidase (ROS production) eliminated the LPS-upregulated cerebrovascular activity. Furthermore, blockage of these ROS and NO pathways reduced the preconditioning effect of LPS. Therefore, LPS may play a more direct role in preventing ischemic damage by increasing blood availability to the affected brain region.

Inflammatory cytokines and the innate immune system

LPS, a component of the gram-negative bacterial cell wall, can illicit a potent innate immune response. While this systemic inflammatory response can be destructive (at doses of 5 mg/kg),⁶⁵ tolerable LPS doses of 0.05 to 1 mg/kg injected intraperitoneally render the brain,¹¹ heart,^66,67 liver,^68,69 kidneys,⁷⁰ and pancreas⁷¹ transiently resistant to subsequent ischemic injury. This preconditioning paradigm relies on the ability of a peripheral signal to cross into multiple organ systems. LPS injected into the gut can signal through peritoneal macrophages and circulating monocytes. Toll-like receptor 4 is a pattern-recognition receptor that binds to pathogen-associated molecular patterns in LPS and initiates a signaling cascade through the NFκB pathway. This pathway culminates in the expression and secretion of several proinflammatory cytokines to fight off the infection and anti-inflammatory cytokines to control the immune response.

The major output of LPS signaling is innate production of proinflammatory cytokines to fight infection and clear cellular debris. Central cytokines, including tumor necrosis factor–alpha (TNFα), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β), can be neurodestructive if administered after ischemia. TNFα administration by cerebroventricular injection after ischemia augmented the extent of injury, and blockage of TNFα signaling proved neuroprotective.^11,72,73 However, in LPS preconditioning, cytokine production precedes ischemia. Intracisternal injections of TNFα before middle cerebral artery occlusion (MCAO) were protective in reducing the infarct size of pretreated mice.⁷⁴ Furthermore, intracisternal injection of ceramide analog, a downstream component of the TNFα signaling pathway, was also capable of reducing the MCAO infarct area.⁷⁵ Preischemic treatment with IL-6 and IL-1 also reduced neuronal damage.^76,77 TNFα knockout mice eliminated the LPS protective phenotype,⁷² demonstrating that cytokine production is a critical feature of LPS preconditioning in ischemia. Additionally, ischemic damage in the absence of LPS preconditioning was exacerbated in TNFα receptor 1 knockout mice.^78,79 Consistently, TNFα protein levels are upregulated after LPS treatment but are downregulated following LPS-preconditioned MCAO.⁷² A unifying theme in LPS preconditioning comprises early activation of the innate immune system with ensuing suppression in ischemia.

As a potential mechanism, the initial inflammatory response induced by LPS appears to render the innate immune system hyporesponsive to subsequent insults such as ischemia. This may occur by persistence of anti-inflammatory cytokines produced by the primary insult. These molecules are expressed in tandem with proinflammatory cytokines to control the innate immune response, but may also play a role in delayed preconditioning. For instance, intravenous or intracerebroventricular IL-10 injection can reduce the infarct size with MCAO.⁸⁰ Alternatively, several proinflammatory cytokine signaling pathways may be downregulated by negative feedback inhibition.^20,81 This inhibition may occur extracellularly, using soluble cytokine receptors, decoy receptors, or receptor antagonists. For example, intravenous injection of IL-1 receptor antagonist can provide neuroprotection against ischemic injury from MCAO.^82,83 Cytokine feedback inhibitors that act intracellularly are also induced with the innate immune response. Intracellular inhibition may involve direct downregulation of cytokine transcription (peroxisome proliferator-activated receptor gamma [PPAR-γ]) or inhibition of intracellular signaling pathways that promote cytokine production (suppressor of cytokine signaling [SOCS] and PI3K). Antisense mRNA knockdown of SOCS-3 exacerbates ischemic injury from MCAO.⁸⁴ The MCAO infarction area is increased after treatment with PPAR-γ antagonists and decreased by PPAR-γ agonists.^85,86 Administration of compounds that increase PI3K signaling is also capable of reducing ischemic damage.⁸⁷ Thus, several defense mechanisms designed to suppress the innate immune response may play an active role in LPS ischemic preconditioning.