Cytochrome P450 CYP2E1 Suppression Ameliorates Cerebral Ischemia Reperfusion Injury

Abstract

Despite existing strong evidence on oxidative markers overproduction following ischemia/reperfusion (I/R), the mechanism by which oxidative enzyme Cytochrome P450-2E1 (CYP2E1) contributes to I/R outcomes is not clear. In this study, we sought to evaluate the functional significance of CYP2E1 in I/R. CYP2E1 KO mice and controls were subjected to middle cerebral artery occlusion (MCAo-90 min) followed by 24 h of reperfusion to induce focal I/R injury as an acute stage model. Then, histological and chemical analyses were conducted to investigate the role of CYP2E1 in lesion volume, oxidative stress, and inflammation exacerbation. Furthermore, the role of CYP2E1 on the blood-brain barrier (BBB) integrity was investigated by measuring 20-hydroxyecosatetraenoic acid (20-HETE) activity, as well as, in vivo BBB transfer rate. Following I/R, the CYP2E1 KO mice exhibited a significantly lower lesion volume, and neurological deficits compared to controls (p < 0.005). Moreover, reactive oxygen species (ROS) production, apoptosis, and neurodegeneration were significantly lower in the CYP2E1(-/-) I/R group (p < 0.001). The BBB damage was significantly lower in CYP2E1(-/-) mice compared to wild-type (WT) (p < 0.001), while 20-HETE production was increased by 41%. Besides, inflammatory cytokines expression and the number of activated microglia were significantly lower in CYP2E1(-/-) mice following I/R. CYP2E1 suppression ameliorates I/R injury and protects BBB integrity by reducing both oxidative stress and inflammation.

Keywords: CYP2E1; blood-brain barrier; inflammation; ischemia/reperfusion; oxidative stress.

Figure 1

Genetic deletion of cytochrome P450-2E1 (CYP2E1) protects against cerebral ischemia/reperfusion (I/R) injury. Representative photomicrographs sets of 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain slices for transient middle cerebral artery occlusion (MCAo) (tMCAo) and permanent MCAo at 24 h are shown in panels (A,B), respectively. Quantitative analysis of total brain infarct volume ratio (in %) is shown in panel (C). Male (M) and female (F) tMCAo (90 min) followed by 22.5 h of reperfusion and permanent ischemia along with aged-match controls were studied separately for lesion volume by TTC staining at 24 h of ischemic insult. Inhibition of CYP2E1 enzyme significantly decreased infarct volume in comparison with wild-type (WT) male. Values are presented as mean ± SD (n = 8–12 per group).

Figure 2

(A) CYP2E1 activity increases following I/R. Mice transient MCAo (tMCAo; 90 min occlusion and 24 h of reperfusion) models are compared with controls for cerebral CYP2E1 activity at 24 h. CYP2E1 activity was assessed by the nitrocatechol method [28]. CYP2E1 activity was increased significantly (61%) following transient MCAo compared with age- and sex-matched controls (n = 7). No significant CYP2E1 activity was found in the CYP2E1(−/−) group. (B). Ninety minutes I/R increases Reactive oxygen species (ROS) production in the brain, as determined by using chloromethyl derivative of dichlorofluorescein diacetate (MC-H₂DCFDA) at 24 h post-reperfusion. The fluorescent signals generated with MC-H₂DCFDA in the brain were increased with the insult of I/R. The analysis of fluorescence intensity illustrates the significant reduction in ROS production in CYP2E1(−/−) mouse after the insult of I/R in comparison to age-matched WT mice. No fluorescence was detected in wells without the incubation of MC-H₂DCFDA. Data are shown as mean + SD. n = 6 male per group.

Figure 3

Apoptosis and neurodegeneration were reduced in CYP2E1(−/−) mice following I/R (tMCAo). (A) apoptosis of mice MCAo brain tissue in each group measured by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) (magnification, ×200) in the MCA territory of the cortex. TUNEL-positive cells (brown staining) significantly decreased in CYP2E1 compared with those in the WT group. TUNEL assay was performed in the cortical cerebral sections. TUNEL-positive cells were counted. Representative figures show CYP2E1 inhibition reduces expression of (B) microglia/macrophage activation (ionized calcium-binding adaptor protein-1 (Iba-1) positive cells), and (C) astrogliosis following tMCAo and 24 h reperfusion (astrocytes glial fibrillary acidic protein (GFAP) positive cells) in the peri-infarct cortex. Astrocytes stained brown.

Figure 4

The quantified level of protein expression of Iba-1 (right) and GFAP (left) in the peri-infarct region of CYP2E1(−/−), CYP2E1(+/+), and control mice brains subject to 90 min MCAo reperfusion are shown in the (top) of the panel. Detection of the Iba-1 and GFAP in mouse peri-infarct areas CYP2E1 and deficient in CYP2E1 (knock-out; KO), was done by Western blot analysis using rabbit polyclonal anti-Iba-1 and anti-GFAP antibody. Data are represented as mean + SD (fold change relative to mean control value), n = (3,5,5) males per group (control, CYP2E1(+/+), and CYP2E1(−/−) for GFAP analysis and n = (3,4,4) males per group for Iba-1 analysis. A statistically significant difference between the expression of both GFAP and Iba-1 protein in CYP2E1(+/+) I/R and CYP2E1(−/−) I/R is observed. Representative Western blots of Iba-1 and GFAP protein levels in the brain of CYP2E1(−/−) I/R, CYP2E1(+/+) I/R, and WT control mice subject to 90 min MCAo are shown at the (bottom) of the panel. β-actin was used as a control for loading.

Figure 5

Inflammatory cytokine protein expression was reduced in CYP2E1(−/−) mice following I/R (tMCAo) insult. Bar graphs show the protein concentration of (A) interleukin (IL)-6, (B) monocyte chemoattractant protein 1 (MCP-1), and (C) tumor necrosis factor (TNF)-α in brain tissues at 24 h of MCAo, measured by ELISA. CYP2E1(−/−) groups show an almost two-fold reduction in inflammatory marker expression following I/R compared to WT I/R. Sham-operated animals did not show a marked increase in IL-6. The levels of brain IL-6, MCP1, and TNF-α were determined by ELISA kits. Data are presented as mean + SD, and n = 6–12.

Figure 6

Ischemia/reperfusion triggers an increase in 20-hydroxyecosatetraenoic acid (20-HETE) synthesis and breakdown of the blood-brain barrier (BBB) in the brain of mice. (A) Representative BBB transfer rate map after I/R in CYP2E1(−/−) and (B) in WT control mice, in vivo MR quantification. Representative structural MRI (above) and corresponding quantitative BBB transfer rate map (below) are shown for three consecutive slices covering most of the ischemic lesion for both CYP2E1(−/−) and WT MCAo. Panel (C) represents a statistical comparison of BBB transfer rates between CYP2E1(−/−) MCAo and WT MCAo as measured by MRI. Furthermore, 20-HETE synthesis following the insult of I/R was increased significantly (p < 0.05) in CYP2E1(−/−) mice (36% of control). However, the 20-HETE synthesis increment in WT- I/R was not statistically significant compare to WT controls (12% of WT control). Data are presented as mean ± SD in panel (D). n = 8–10. Mouse MCAo was subjected to 90 min MCA occlusion and 24 h of reperfusion. In this CYP2E1 (−/−) model, in comparison to WT controls, lesion volume was significantly lower. Interestingly, there was no significant damage to BBB as measured by the rate of leakage of the contrast agent (Gd-DTPA) from the blood into cerebral tissues through BBB. Anatomical images acquired with RARE (Rapid Acquisition with Relaxation Enhancement) T2w MRI sequences. The BBB transfer rate maps were constructed from perfusion data acquired by using a dynamic contrast-enhanced MRI technique (DCE-MRI) with Gd-DTPA bolus injection. Images acquired by a 7T research-dedicated Bruker magnet equipped with a cryogenic quadrature RF surface coil as the transmitter/receiver.