Brain ischemia may be either widespread or localised. A major portion of the brain or the whole brain may be affected by global cerebral ischemia which can be result from systemic processes like shock and cardiac collapse. Long-term global ischemia may cause brain damage that is irreversible [1]. Contrarily, arterial blood flow restriction most often results from thrombosis or embolism of a major cerebral artery, usually the middle cerebral artery [2] resulting in focal ischemia. The goal of treating ischemic stroke is to salvage as much of the ischemic penumbra as early as feasible. About 50% of all patients with acute ischemic stroke on MRI still had viable penumbras [3]. It is well-known that reperfusion after brain ischemia leads to secondary injury characterized by functional and structural injury causing paradoxical neuronal dysfunction and necrosis. This perfusion damage is complex and multifactorial but it is mingled with a fulminant infiltration of leukocytes, and consecutive release of inflammatory mediators and free radicals into the ischemic tissue [4]. After reperfusion, the blood-brain barrier is disrupted, causing edema, more neuroinflammation and free radical damage, and hemorrhagic transformation of the ischemic stroke that worsen the prognosis of the initial stroke [5]. Although full recanalization is the goal of mechanical and pharmaceutical revascularization treatments, downstream reperfusion is not always accomplished resulting in the no-reflow phenomena [6]. Despite clot removal, it is thought that microvascular blockage, reduced vasomotor control, decreased nitric oxide generation, and consequent vasoconstriction is likely the cause of the defeat to re-perfuse tissue [7].

Perindopril is a non-sulfhydryl prodrug own to angiotensin-converting enzyme (ACE) inhibitor family. It is rapidly metabolized to perindoprilat, its active form which is competitive inhibitor of angiotensin converting enzyme responsible for conversion of angiotensin I to angiotensin II. Subsequently, angiotensin II-mediated vasoconstriction and angiotensin II-stimulated aldosterone secretion are suppressed [8]. Coenzyme Q10 is a potent antioxidant and free radical scavenger and it also has anti-inflammatory effect. It is found in oxidized (ubiquinone) or reduced (ubiquinol) form. It is a necessary cofactor in mitochondrial electron transport system and oxidative phosphorylation to produce adenosine triphosphate (ATP) [9, 10].

The study was conducted at the Kufa Faculty of Medicine’s Department of Pharmacology. After being taken from the animal house at Kufa College of Science, a total of forty-nine adult male Sprague-dawley rats weighing between 200 and 300g were housed in the same location in a temperature-controlled room (25\(\pm\)1oC) with 60-65% humidity. The lighting was kept on a 12-hour cycle of light and dark. Food and water were given to the rats at no cost (ad libitum). The institutional animal care and use committee of Kufa University has approved the study and matched it with laboratory animal guide care. The rats were divided into seven groups at random, with seven rats in each group; Group 1 (Sham group): Rats undergo the same surgical treatments as other rats, but without BCCAO. Group 2 (Control group): Rats prone to BCCAO for 30 minutes, followed by an hour of reperfusion. Group 3 (vehicle group for carboxymethylcellulose): Seven days prior to surgery, the rats received the same volume of oral 1% carboxymethylcellulose. After that, they received 30 minutes of BCCAO and an hour of reperfusion. Group 4 (Distilled water vehicle group): Seven days prior to surgery, the rats received oral distilled water. After that, they received BCCAO for thirty minutes and reperfusion for an hour. Group 5: (Perindopril-treated group): Seven days before to surgery, the rats received 4 mg/kg/day of perindopril via a gastric gavage tube. They were then given BCCAO for 30 minutes and reperfusion for an hour. Group 6 (CoQ10-treated group): Seven days prior to surgery, the rats received 40 mg/kg/day of CoQ10 by gastric gavage tube. These rats subsequently had 30 minutes of BCCAO and an hour of reperfusion. Group 7: Perindopril and CoQ10 Combination: Using a gastric gavage tube, rats received 4 mg/kg/day of perindopril and 40 mg/kg/day of CoQ10 for 7 days prior to surgery. After 30 minutes and an hour after reperfusion, they were given BCCAO.

Induction of global cerebral ischemia

Under general anesthesia using intraperitoneal injections of xylazine (10 mg/kg) and ketamine (100 mg/kg). Both common carotid arteries were exposed through a median neck incision, and they were occluded for half an hour to cause ischemia. After the clamps were removed, reperfusion was allowed to continue for an hour [10].

Preparation of brain tissue samples for histopathology

Hematoxylin and eosin staining was applied after the brain tissues were fixed in 10% formalin and placed in an automated tissue processor. Histological observations yielded a score of [11]; 0 (Normal) indicates no damage. Three categories are available: 1 (mild): minimal bleeding, 2 (moderate): modest interstitial edema or eosinophilic neurons, and 3 (severe): local necrosis. Cerebral infarct volume was assessed by immersion method of TTC stain [12]. Cerebral levels of NF-\(\kappa\)B was analyzed using Dako EnVision IHC technique [13].

Statistical analysis

Data analysis was done using SPSS version 26. The Shapiro-Wilk test was used to determine if the data were normal. The LSD post hoc test was used to analyze the parametric data after the ANOVA test. Kruskal-Wallis and post hoc tests are used to evaluate non-parametric testing. A P-value of less than 0.05 was considered statistically significant.

In our study, cerebral IL-6, TNF-\(\alpha\), and ICAM-1 were significantly increased (p<0.05) in control and vehicle related to sham groups. These increments were markedly decreased (p<0.05) in the combination treatment group compared with control, both vehicles, perindopril-, and CoQ10-treated groups (Figures 1, 2, 3 and 4). The ICAM-1 reduction in the combination group was more significant than perindopril- and CoQ10-treated groups, while IL-6 reduction by the combination treatment was more significant than CoQ10-treated group only. The other decrements among the treatment groups were insignificant.

Figure 4: IL-10 (pg/ml) Among Groups

The cerebral T-AOC level were markedly reduced (p<0.05) in control and vehicle related to sham groups. The T-AOC level was significantly increased (p<0.05) by combination treatment group in respect to control and vehicle groups (Figure 5). Interestingly, the increment in the combination treatment group was more significant (p<0.05) than that of perindopril-treated group, while this rise was insignificant related to that of CoQ1-treated group.

Figure 5: T-AOC (U/ml) Among gGroups

In the current study, NF-\(\kappa\)B p65 nuclear expression was considerably elevated (p<0.05) in control and vehicle groups compared with sham group. This considerable elevation was markedly decreased (p<0.05) in the combination group. There were insignificant differences among the three treatment groups and also the sham group (Figure 6).

Figure 6: IHC of NF-\(\kappa\)B Nuclear Expression in Rat Brain. (400X)

Effects of combined perindopril and CoQ10 treatment on cerebral histopathology: The histopathological assessment demonstated normal histology in all sham group. The control and vehicle rats showed considerably severe (p<0.05) ischemic injury. The combination treatment group revealed marked decrement (p<0.05) in these findings. There were insignificant differences among the three treatment groups (Figure 7).

Figure 7: Cross Section of Rat Cerebrum Stained with Hematoxylin and Eosin

The present work revealed that there was a significant elevation (p<0.05) in the cerebral infarct size percentage in control and vehicle in relation to sham groups. This percentage was markedly decreased (p<0.05) in the combined treatment group (Figure 8 and 9). Interestingly, this decrement was more marked (p<0.05) in the combined treatment group than CoQ10-treated group, while was insignificant between combined treatment and perindopril-treated group.

Figure 9: Photogrphs of Coronal Brain Slices Stained with TTC

Stroke can generate immune responses that lead to activation of inflammatory cell and infiltration. Reperfusion of blocked vessel can cause generation of ROS that activate ischemic cells to release inflammatory mediators causing upregulation of adhesion molecule and recruitment of leukocytes, that can secrete more cytokines and ROS. These substances may induce blood-brain barrier disruption and more neuronal damage [14].

Effects of combined perindopril and CoQ10 treatment on cerebral IL-6, IL-10, TNF-\(\alpha\), ICAM-1, T-AOC, and NF-\(\kappa\)B p65: Perindopril, a centrally active angiotensin converting enzyme inhibitor, can pass blood-brain barrier very easily. Perindopril provides a neuroprotective property against cerebral ischemia stemmed from its antioxidant and free radical scavenger actions, and also nitric oxide release [15]. Angiotensin-converting enzyme inhibitors reduce the plasma inflammatory cytokines levels [16]. Perindopril also can reduce pro-inflammatory/pro-oxidant Angiotensin-II while increase anti-inflammatory/antioxidant Angiotensin-1-7 in methotrexate-induced intestinal injury in rats [17]. [18] studied perindopril effect and found that perindopril pretreatment, in rats with hepatic ischemia reperfusion injury, markedly reduced (p<0.05) both serum TNF-\(\alpha\) levels and hepatic NF-\(\kappa\)B p65 mRNA gene expression, while IL-10 serum level was significantly increased compared to ischemia-reperfusion group. In addition, perindopril caused considerable reduction of markers of hepatic oxidative stress like malondialdehyde and superoxide dismutase related to ischemia-reperfusion group.

The CI/RI may induce oxidative stress and low levels of protective antioxidant such as CoQ10 in the brain [19]. As a result, adequate CoQ10 levels may play a protective role against oxidative stress and inflammation in brain ischemia [20]. [21] showed that levels of hippocampal TNF-\(\alpha\), ICAM-1, and also expression of NF-B p65 were remarkably decreased in CoQ10-pretreated rats related to control group with transient BCCAO.

For best of our knowledge, there are no studies on combined perindopril and CoQ10 treatment effects on cerebral IL-6, IL-10, TNF-\(\alpha\), ICAM-1, T-AOC, and NF-\(\kappa\)B levels in rats exposed to CI/RI.

Effects of perindopril and CoQ10 combination treatment on cerebral histopathology: Perindopril and CoQ10 combination treatment showed a significant amelioration in the ischemic changes. [15] found that histopathological damage was considerably lower in perindopril pretreatment rats with permanent cerebral ischemia than that of ischemic group. [22] found substantial reduction in the ischemic changes in CoQ10-pretreated diabetic rats in respect to control rats with reversible middle cerebral artery occlusion.

As far as we know, there are no studies on effects of perindopril and CoQ10 combination treatment on cerebral histopathology in rats exposed to CI/RI.

Effects of perindopril and CoQ10 combination treatment on cerebral infarct size [23, 24, 25] showed that brain infarction size measured by TTC stain was significantly reduced in CoQ10-pretreated group related to control group with acute reversible middle cerebral artery occlusion. As far as we can tell, there is no studies on effects of perindopril or combined perindopril and CoQ10 treatment on infarct size in rats with CI/RI.

The present study demonstrated that perindopril and CoQ10 combination treatment can significantly decrease cerebral infarct size and ameliorates histopathological damage in male rats exposed to CI/RI. The neuroprotective properties of perindopril and CoQ10 possibly derived from their anti-inflammatory and antioxidative effects.

This research paper received no external funding.

The authors declare no conflicts of interest.

All authors contributed equally to this paper. They have all read and approved the final version.

Informed consent was obtained from all participates in the study as needed.

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