GA administered during brain development caused significant upregulation of ROS, which was accompanied by considerable membrane lipid peroxidation, mitochondria damage, and neuronal loss in the subiculi of young rats. ROS scavenging with EUK-134 or protection of mitochondria with R(+) PPX around the time of anesthesia exposure resulted in significant downregulation of ROS and lipid peroxidation, as well as prevention of mitochondrial morphological damage, protection of neuropil, and prevention of neuronal loss. Most importantly, peri-anesthesia treatment with EUK-134 or R(+) PPX prevented anesthesia-induced cognitive impairment.
Morphologically distorted and functionally impaired mitochondria generate excessive ROS and reduce ATP production, thereby damaging neurons (Reddy, 2006
). Our earlier in-vivo findings indicated that GA increases mitochondria membrane permeability in part by downregulating bcl-xL
protein levels, thus causing cytochrome-c leakage and caspase-cascade activation (Yon et al., 2005
). Increasing evidence suggests that ROS have a key role in promoting cytochrome-c release from mitochondria (Nishimura et al., 2001
; Petrosillo et al., 2011
; Galindo et al., 2003
), indicating that GA-induced cytochrome-c leakage can also be caused by excessive GA-induced ROS upregulation. Although the temporal relationship between ROS upregulation and cytochrome c leak remains to be established it appears that a crucial (and perhaps the initial) cellular target of GA-induced developmental neurodegeneration is immature mitochondria, which seem to be damaged by multiple means converging to induce long-lasting detrimental effects on developing neurons.
Mitochondrial dysfunction and excessive ROS production, accompanied by significant peroxidation of cellular and subcellular lipid membranes, are important in the development and progression of several neuronal diseases marked, among other symptoms and signs, by severe cognitive decline (Reddy, 2006
; Trushina et al., 2004
; Bennett, 2005
). Neurons are highly dependent on glucose for ATP synthesis and produce ROS as byproducts of oxidative phosphorylation within mitochondria. At the same time, neurons, because of their high oxygen requirements and relative deficiency in oxidative defenses (in particular, low to moderate activity of catalase and Mn-SOD), are highly sensitive to excessive ROS production. This vulnerability, combined with their high content of polyunsaturated fatty acids, makes them susceptible to excessive lipid peroxidation and cellular damage (Halliwell, 1992
). Here we have presented evidence that early exposure to GA makes developing neurons susceptible to ROS-induced mitochondria-propagated lipid peroxidation that may be consequential for the observed neuronal injury and impairment of proper cognitive development. Based on significant prevention of GA-induced pathomorphological and cognitive impairments by EUK-134 and R(+) PPX, therapeutic interventions aimed at decreasing ROS production, preventing excessive lipid peroxidation and protecting mitochondria could be the key to safe use of GA during early stages of brain development. This is of particular interest considering the fact that the exposure to GA is often a planned event where a safening agent could be co-administered in timely fashion. Such neuroprotective strategies should enable the use of general anesthetics to their full therapeutic potential while potentially avoiding long-term neuronal damage and/or cognitive sequellae.
The possibility that GA is a direct ROS producer cannot be excluded. For example, N2
O, which is one of the anesthetics used in our regimen can generate free oxygen (most likely hydroxy) radicals in the presence of trace metals such as iron and copper (Orestes et al., 2011
), both commonly found in biological fluids and neurons. Upregulation of ROS promotes metal-catalyzed protein oxidation, which in turn permanently alters the function of various cellular proteins (Stadtman and Berlett, 1991
; Stadtman, 1993
). For example, N2
O causes inhibition of low-voltage-activated calcium channels by oxidizing critical amino acids in the functional pore of the channel (Orestes et al., 2011
). However, having considered that possibility we believe that it is unlikely that N2
O production of ROS and potential protein oxidation is the main culprit for developmental neurotoxicity we present in this study. For example, our previous work using N2
O as a sole anesthetic in clinically relevant concentrations (up to 180-vol%) has shown no detrimental effects on neuronal integrity nor any signs of neuronal apoptosis during early stages of brain development (Jevtovic-Todorovic et al., 2003
). Nevertheless, some degree of N2
O-induced ROS generation, combined with mitochondria-induced ROS production resulting from GA-induced mitochondrial impairment may overwhelm the scavenging system, initiating the cascade of events in which mitochondria becomes not only a target, but also a culprit.
Two agents that we have chosen to study, EUK-134 and R(+) PPX, have distinct properties. EUK-134, a synthetic ROS scavenger with both Mn-SOD and catalase activity, catalytically eliminates both superoxide and hydrogen peroxide (Gonzales et al., 1995
), but does not directly protect mitochondria. It is a potent and stable agent with a high degree of bioavailability which has been shown to offer neuroprotection against excessive ROS production in several in-vivo (Baker et al., 1998
; Rong et al., 1999
; Tiwari et al., 2009
) and in-vitro (Fonck et al., 2003
; Pong et al., 2002
) models of neuronal injury. Here, for the first time, we show that EUK-134 decreases GA-induced ROS upregulation and, most importantly, blocks GA-induced learning impairment during development.
Whereas EUK-134 mainly scavenges ROS in the cytoplasm, thus indirectly protecting mitochondria and other organelles, R(+) PPX is a direct protector of mitochondrial integrity that scavenges superoxide anions inside the organelle. It easily crosses the blood-brain barrier, concentrates in the brain at about a 6-fold higher concentration than it does in plasma, and is taken up by mitochondria, where it is concentrated more than 8-fold (Danzeisen et al, 2006
). Of particular interest here is that R(+) PPX, as opposed to S(−) PPX, has minimal dopaminergic activity. Accordingly, it has no apparent toxicity or significant side effects at very high doses. For example, the LD50
for i.p. R(+) PPX in rodents is ~375 mg/kg (unpublished observation), which is more than 80-fold higher than the combined four doses used in our protocol to abolish GA-induced cognitive impairment. Importantly, R(+) PPX has been used in clinical trials and has been shown to slow the progression of clinically documented amyotrophic lateral sclerosis without causing harmful side effects (Wang et al., 2008
Since proper function of developing neurons relies on a fine balance between ROS production and elimination, the wellbeing of mitochondria and proper function of the scavenging system are crucially important. Consequently, protective interventions based on tight control of ROS production and/or elimination could be the earliest defenses against the progression of mitochondria damage to further ROS production, ATP depletion, caspase activation, and DNA fragmentation. Although the net effects of EUK-134 and R(+) PPX are ROS downregulation and curtailing of lipid peroxidation, those effects could be considered direct in the case of the EUK-134 and perhaps indirect in the case of R(+) PPX. In using these two agents, our goal was to examine the temporal role of ROS scavenging versus the protection of mitochondria integrity. We hypothesized that if EUK-134, a Mn-SOD/catalase mimetic, proved to be more effective in alleviating GA-induced developmental neurotoxicity, then GA’s initial attack could be aimed at the complex scavenging system. By promoting effective and timely ROS elimination, EUK-134 treatment blocks ROS upregulation and ROS-induced mitochondria damage thus preventing further ROS upregulation. On the other hand, if R(+) PPX was shown to be more effective, the initial damage could be due to mitochondria dysfunction. Since both treatments were very effective when administered at the time of GA exposure, it may well be that GA-induced developmental neurotoxicity is a complex combination of extensive ROS production and impaired ROS scavenging that are tightly intertwined. Indeed, Guo et al. (2008)
have proposed a “double jeopardy” in leptin-deficiency models of diabetes where the worsening of mitochondrial function leads to enhanced ROS production in tandem with impaired expression or function of scavenging machinery.
Although the focus of our study was on downstream consequences of mitochondrial impairment and upregulation in ROS it is noteworthy that the upstream trigger of a vicious cascade of mitochondrial damage could be the excessive release of calcium from the endoplasmic reticuli (EM) resulting in intacytosolic and mitochondrial calcium overload. This in turn may cause cytochrome c leak (Hanson et al., 2004
) which could further promote mitochondrial dysfunction. Indeed, Zhao and colleagues (2010)
have shown that by modulating inositol 1,4,5-trisphosphate receptors, isoflurane induces significant calcium release from the ER resulting in acute elevation of cytosolic calcium and modulation of mitochondrial bcl-xL
protein levels which in turn promoted apoptotic neuronal death in the immature rat brain. It remains to be established whether and how anesthesia-induced modulation of calcium homeostasis is affected by EUK-134 or R(+) PPX-co-administration.
Based on our findings, determination of a temporal relationship between GA exposure, ROS upregulation, and mitochondrial damage appears to have only theoretical importance, despite the logical notion that the best intervention should be one specific to preventing the very initial steps. If, for instance, mitochondria are the initial GA target, agents aimed at protecting mitochondria, such as R(+) PPX, would be the most selective therapeutic approach. If, however, ROS accumulation is the initial event, then ROS scavengers should be the first line of defense, so that ROS-induced mitochondria damage is never initiated. Nevertheless, it appears that peri-anesthesia exposure to a “safening agent,” whether aimed at curtailing ROS production or promoting ROS elimination, offers equally impressive prevention against the multifaceted impairments caused by early exposure to GA. Thus, our work suggests that GA-induced developmental neurotoxicity is the result of a highly complex interaction between ROS-induced, mitochondria -propagated, and mitochondria-induced ROS-propagated cascades of events that ultimately lead to neuronal damage and behavioral impairment.
Protective strategies based on preserving mitochondrial integrity have been previously shown to provide significant downregulation of GA-induced developmental neurodegeneration. For example, both melatonin, a naturally occurring sleep hormone that upregulates bcl-xL (Yon et al., 2006
), and carnitine, a nutritional supplement that protects mitochondria integrity (Zou et al., 2008
) cause significant, although not complete, neuronal protection. However, neither agent is known to protect against GA-induced cognitive impairment.
We present evidence that GA-induced long-term cognitive impairments in immature rats are prevented by two agents, EUK-134 and R(+) PPX, both of which prevent excessive ROS upregulation. Deciphering the earliest and most vulnerable cellular targets and their relative importance in promoting long-term cognitive impairments is the key to effective intervention. This study points toward an attainable means of providing safe anesthesia to the youngest members of our society.
Early exposure to general anesthesia causes developmental neuroapoptosis in the mammalian brain and long-term cognitive impairment that is caused, at least in part, by mitochondrial damage and excessive free oxygen radical production. Consequently, timely mitochondrial protection and free oxygen scavenging prevents anesthesia-induced lipid peroxidation, preserves mitochondrial integrity, and prevents neuronal loss. Importantly, it completely prevents anesthesia-induced cognitive impairment.