Cytochrome P450 CYP2E1 Suppression Ameliorates Cerebral Ischemia Reperfusion Injury

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.

measured in Contralateral and Ipsilateral of focal ischemia during occlusion and post reperfusion respectively. Data are represented as mean ± Standard deviation (SD). Three randomly experimental groups; Sham operated mice (n=5), CYP2E1(-/-) mice (n=7), and WT mice (n=7) were used to generate the data.
TTC staining-Slices were then incubated in a 2% solution of TTC in 0.1 M PBS (pH 7.4) at 37°C for 30 min and fixed in 10% formalin. Normal brain tissue was stained red, and the infarct tissue was white. TTC-stained brain sections were photographed using a digital camera (Powershot 400 digital camera, Canon). The infarct size was calculated by the researcher (ST) blind to the group identity, and the percentage of the infarct area with respect to the total area was digitally quantified by ImageJ. The infarct volume percentage was expressed by the sum of the infarct area of each section / the sum of the area of each section.
Preparation of brain membranes-Brains were pooled in 10 volumes of cold homogenization buffer (100 mM Tris (pH 7.6 at 4°C) with 0.1 mM EDTA, 0.32 M sucrose and 0.1 mM DTT) and manually homogenized on ice using a glass homogenizer. Homogenates were centrifuged twice at 3000 × g for 10 min to remove cellular and nuclear debris. Next, we took supernatants (S1) and spin (Beckman Optima TL centrifuge; TLA45 rotor) at 110,000 × g at 4°C for 100 min to yield crude cytosol (S2) and crude membrane pellet (P2). Then we resuspended pellet in homogenization buffer and spin again at ~110,000 x g to yield washed crude membrane pellet (P2'). Protein concentration was measured by BCA protein assay kit (Thermo Fisher Scientific). For immunoblotting, the resulting membrane pellets were resuspended in a storage solution containing 100 mM Tris (pH 7.4), 0.1 mM EDTA, 0.1 mM DTT, 1.15% (w/v) KCl, and 20% (v/v) glycerol, and then samples were stored in aliquots at −80°C.
3 CYP2E1 enzyme activity measurement-Briefly, a mixture containing 440 μL assay buffer of 100 mM potassium phosphate to PH 7.4, 10μL of 5 mM p-nitrophenol, and 25μL of 20 mM NADPH was incubated for 15 min. After that, samples (P2', 200μg protein) were added and incubated at 37°C for 60 min in a dark room. The reaction then terminated by adding 100μL of 20% trichloroacetic acid to the suspension. The samples were placed on ice for 2 min. After centrifugation at 10,000 × g for 5 min, 500μL of supernatant mixed with 250μL of 2 M NaOH. The absorbance of p-nitrocatechol then determined at 535nm in a multiplate reader spectrophotometer (Fluostar Optima, BMG Labtech). Activity expressed as picomoles formed per hour per micrograms of cell protein (Figure 2).

Immunohistochemistry protocols and antibodies for TUNEL assay-
To assess the extent of brain damage, paraffin embedded coronal brain section processed for TUNEL assay. TUNEL staining was performed according to the manufacturer's instruction (Apop tag R , S7100, Milipore Sigma USA). Briefly, the brain tissue was placed in 4% paraformaldehyde and embedded in paraffin sectioned. After washing, the sections were incubated in TDT enzyme at 37°C for one hour in a humidified chamber. The reaction was revealed with 0.06%. TUNEL signal was detected with Alexa 488-labelled secondary antibody for streptavidin. Cell nuclei were stained with DAPI.
Negative controls of TUNEL staining were performed by omitting TdT. The number of apoptotic/necrotic neurons was determined by counting the NeuN/TUNEL double-stained cells in the infarct region. The percentage of TUNEL-positive cell in total DAPI positive nuclear cells was presented.
Immunohistochemistry and Western Blotting-Relative levels of Iba-1, GFAP, and β-actin in the supernatant fraction from the brain extract were determined by western blot analysis (rabbit 4 polyclonal antibodies: Iba-1 (ab-153696); GFAP (sc-6170), β-actin (sc-130657), Abcam Cambridge UK and Santa Cruz Biotechnology, Santa Cruz, CA), as described previously [21]. Relative intensities of western blot bands were assessed by densitometry in triplicate for each sample. Densitometric analysis was done using IQTL software (GE Life Science, Piscataway, NJ). Animal imaging conditions-Isoflurane gas was used for anesthesia the same way as it was used for surgery with a slightly different dosage to minimize the effect of anesthesia on CBF (induction dosage 2-2.5% and maintenance dosage 1.5%-2%; controlled for a stable respiration rate), at 1 L/min compressed air flow under spontaneous respiration throughout the entire course of each imaging session. Real time monitoring of physiological parameters (heart rate, respiratory rate, and body temperature) were conducted during each imaging session for signs of distress (Small Animal Instruments Inc., Stony Brook, NY). MR imaging protocol for each session was included sequences for anatomical and DCE-MRI.

Figure S2. Neurological deficit following I/R insult-The neurological deficits were
assessed and scored on a 5-point scale based on the report of Longa et al [13]. A statistically significant differences between Wild Type (WT) MCAo group and CYP2E1 KO MCAo group was observed (n=12, p<0.001).