The abolishment of anesthesia-induced cognitive impairment by timely protection of mitochondria in the developing rat brain: the importance of free oxygen radicals and mitochondrial integrity

Abstract

Early exposure to general anesthesia (GA) causes developmental neuroapoptosis in the mammalian brain and long-term cognitive impairment. Recent evidence suggests that GA also causes functional and morphological impairment of the immature neuronal mitochondria. Injured mitochondria could be a significant source of reactive oxygen species (ROS), which, if not scavenged in timely fashion, may cause excessive lipid peroxidation and damage of cellular membranes. We examined whether early exposure to GA results in ROS upregulation and whether mitochondrial protection and ROS scavenging prevent GA-induced pathomorphological and behavioral impairments. We exposed 7-day-old rats to GA with or without either EUK-134, a synthetic ROS scavenger, or R(+) pramipexole (PPX), a synthetic aminobenzothiazol derivative that restores mitochondrial integrity. We found that GA causes extensive ROS upregulation and lipid peroxidation, as well as mitochondrial injury and neuronal loss in the subiculum. As compared to rats given only GA, those also given PPX or EUK-134 had significantly downregulated lipid peroxidation, preserved mitochondrial integrity, and significantly less neuronal loss. The subiculum is highly intertwined with the hippocampal CA1 region, anterior thalamic nuclei, and both entorhinal and cingulate cortices; hence, it is important in cognitive development. We found that PPX or EUK-134 co-treatment completely prevented GA-induced cognitive impairment. Because mitochondria are vulnerable to GA-induced developmental neurotoxicity, they could be an important therapeutic target for adjuvant therapy aimed at improving the safety of commonly used GAs.

Figure 1. Anesthesia causes significant ROS upregulation in immature subiculi that is ameliorated by a synthetic scavenger, EUK-134

A. Subicular sagittal slices from sham control PND7 rats (A) displayed only a few H₂-DCFDA-positive pyramidal neurons (pyramidal layer is roughly outlined). B. There were abundant H₂-DCFDA-positive profiles (green fluorescence) in the pyramidal layer of GA-treated animals. C. EUK-134 co-treatment reduced GA-induced up-regulation of ROS. D. Quantification of ROS-positive pyramidal neurons showed significant upregulation in GA-treated subiculi compared to sham controls (***, p < 0.001). This was significantly diminished by EUK-134 co-treatment (~ 5-fold decrease compared to GA-treated animals; **, p < 0.01). Consequently, ROS labeling was not significantly higher in GA+EUK-treated animals than in shams (n = 4 rat pups per group).

Figure 2. Significant anesthesia-induced lipid peroxidation in immature subiculi was reduced by EUK-134 co-treatment

Quantification of pg 8-isoprostane, the most abundant prostaglandin-like compound formed in vivo from the free-radical-catalyzed peroxidation of essential fatty acids (primarily arachidonic acid) showed significant upregulation in GA-treated rats compared to sham control (*, p < 0.05) and EUK-alone groups (**, p < 0.01). Co-treatment with EUK significantly downregulated pg 8-isoprostane production compared to that in the GA-alone group (over 2-fold) (***, p < 0.001) (n = 5–6 rat pups per group).

Figure 3. EUK-134 or R(+) PPX co-treatment prevented the significant morphological damage to subicular mitochondria caused by anesthesia

A. Neuropil of GA-treated subiculi analyzed 14 days after anesthesia (at PND 21) contained many swollen mitochondria with balloon-like cristae. Left Enlargement: The inner mitochondrial membrane appeared disintegrated, giving the mitochondrial matrix a disorganized and swollen look (early stage, open arrows). Right enlargement: Many mitochondrial profiles appear dark and condensed without a clear outline between the inner and outer membranes (late stage; closed arrows in left and right panels). B. Mitochondrial profiles in GA + EUK-treated and C. GA + PPX-treated subiculi show nicely stacked cristae with intact inner and outer membranes. D. Mitochondrial density analyses revealed significant increase in the percentage of abnormal-looking mitochondria in GA-treated subicular neuropil compared to sham controls (p< 0.001). Co-treatment with either EUK-134 or PPX resulted in complete mitochondrial protection as indicated by a significant decrease in the density of abnormal-looking mitochondria (p< 0.001) when compared to GA-treated group. The protection was of such degree that GA+EUK and GA+PPX groups were practically indistinguishable from sham controls (n=3 rats per each treatment group).

Figure 4. Significant anesthesia-induced disturbance of neuropil architecture in subiculi was prevented by EUK-134 or R(+) PPX co-treatment

A. Normal-looking sham control neuropil with tightly packed neuronal and glial profiles. B. The neuropil of GA-treated rat appears strikingly disorganized with numerous swollen neuron-glial profiles. There is an abundance of degenerated mitochondria in the neuropil (closed arrows). Many disorganized microtubules seem bunched in the periphery of neuron-glia profiles (asterisks). Due to extensive swelling, the microtubules often had vacuolar appearance (open arrows). C. Neuropil in GA + EUK- 134 treated and D. GA + PPX-treated groups were indistinguishable from sham control neuropil.

Figure 5. Significant anesthesia-induced neuronal deletion in subiculi was prevented by EUK-134 or R(+) PPX co-treatment

In PND 53 rats, there was significant (40%–50%) neuronal loss in GA-treated animals as compared to sham controls (**, p < 0.01). Treatment with either EUK-134 or PPX around the time of GA exposure prevented neuronal loss; i.e., the GA+EUK and GA+PPX groups had neuronal densities similar to those in sham controls and significantly higher than those in rats treated with GA alone (**, p< 0.01). (n = 4–6 animals per group).

Figure 6. Anesthesia exposure did not impair rats’ nutritional development

There were no differences in daily weight gain between treatment groups. Food restriction, a necessary component of the RAM learning protocol, caused no more than 10%–15% loss in body weight loss.

Figure 7. Significant cognitive impairment induced by early exposure to anesthesia was completely prevented by EUK-134 or R(+) PPX co-treatment

A. On the 8-arm RAM test, GA-treated rats were significantly impaired relative to sham controls (***, p < 0.001) in terms of days required to reach a criterion demonstrating learning (8 out of 9 correct responses for 4 consecutive days). B. The GA + EUK-treated group had a significant decrease in days required to reach criterion as compared to the GA-treated group (***, p < 0.001). There was no difference in learning when the GA+EUK group was compared to sham controls. C. The GA+PPX-treated group showed significant improvement in learning as compared to GA-treated group (***, p < 0.001). Comparison of the GA+PPX group to sham controls showed no difference in learning. The learning abilities of EUK-134- (B) and PPX-alone (C) groups were similar to sham controls (A) (n= 19–35 animals per group).

Figure 8. EUK-134 or R(+) PPX co-treatment prevented the slowing of learning acquisition induced by early anesthesia exposure

A. The acquisition rate of the GA-treated group (closed squares) began to slow as compared to that of sham controls (closed triangles) by the fourth block of trials and remained substantially slower for the remainder of training. GA + EUK- (closed circle) and GA + PPX-treated (closed inverted triangles) groups initially exhibited slightly faster acquisition than did the sham control group; after the 5^th block of trials, their learning curve closely trailed that of sham controls. Five out of 35 (~ 15%) GA-treats rats did not learn the task during the 21 days allotted; all sham controls completed the task several days earlier. In the GA + PPX- group, all 20 animals completed the task. In the GA + EUK-134 group, 2 of 26 rats (~ 7%) did not learn the task. B. By plotting the cumulative scores for the GA-treated group relative to those for other treatment groups, we found that while only 51% of GA-treated rats had reached the criterion, 90% of sham controls had done so. In addition, when only 79% of GA-treated rats had reached criterion, all sham controls and GA+PPX animals as well as 93% of GA+EUK animals had mastered the task. Either EUK-134 or PPX co-treatment closed the gap (shaded area) in learning abilities between GA-treated (lower dotted line) and sham-control animals (upper dotted line) (n = 19–35 animals per group).