Postconditioning with D-limonene exerts neuroprotection in rats via enhancing mitochondrial activity

Animals

In the present study, 72 Sprague-Dawley rats weighing 250–300 g were purchased from the animal center and housed in a 12 h cycle of light/dark at a typical animal housing temperature of around 24 °C and humidity of 50 %. All experiments and animal handling techniques were carried out with the agreement of the university Committee for Animal Ethics (no. 2022-AE305).

Cerebral I–R injury induction and study protocol

According to the previous studies, the middle cerebral artery occlusion (MCAO) model was used to develop cerebral I–R injury. The rats underwent an intraperitoneal chloral hydrate anesthesia (400 mg/kg). Then, the right carotid artery was exposed and a rounded filamentous thread with a diameter of 0.234 mm was conducted into the internal carotid from side of the external carotid trunk. The thread was slightly pushed forwardly for a length of 18–20 mm to block the MCA origin. After being left in place for 2 h (ischemic time), the thread was removed to begin reperfusion. During the course of the procedure, a heating pad was used to maintain a body temperature between 37 and 37.5 °C. The animals were anesthetized again 24 h after reperfusion and killed for sampling. In transient focal cerebral ischemia models, MCAO model is typically induced for durations of 60, 90, or 120 min. Various extent of resulting infarct volumes are measured in ipsilateral hemisphere after 24 h of reperfusion [16]. We chose 120 min ischemia and 24 h reperfusion. Rats in sham experienced the same procedures except for MCAO induction. Rats that had extensive bleeding or premature death during surgery were excluded from the study and sampling.

The grouping of animals for this experiment was done according to the following category: (1) Sham, (2) IR, (3) IR+LIM10, (4) IR+LIM100, (5) IR+5HD, and (6) IR+5HD+LIM100. Each group had six rats for each of the infarct volume determination or biochemical and molecular evaluations. In groups of rats receiving LIM, 10 mg/kg (LIM10) or 100 mg/kg of limonene (LIM100), dissolved in 1 % DMSO, was injected by intraperitoneal (i.p.) injection 5 min prior to reperfusion. Other groups received the same amount of 1 % DMSO, i.p. Also, the selective inhibitor of mK–ATP channels, 5-hydroxydecanoate (5HD), was prepared at a concentration of five μM and administered to the corresponding groups, 10 min before injecting 100 mg/kg LIM. Accurate determination of the effective dose of LIM in I–R studies has not yet been performed. In some studies of experimental brain damage in rats, different doses of the drug have been tested [17]. In one study, LIM 100 mg/kg was effective in protecting the brain against dementia [18]. While in another study, 10 mg/kg of it had a significant effect on improving the memory of rats [15]. However, there are reports of side effects such as organ toxicity and increased oxidative stress at higher doses [17]. Because of these findings, two doses of 10 and 100 mg/kg LIM, as the effective doses in previous reports, were chosen to evaluate their effects on cerebral I–R injury in the present study.

Grading of neurological deficits

Neurological deficits grading setting was performed 30 min after occlusion onset to confirm successful development of MCAO, and 24 h after occlusion immediately before euthanizing the rat. This setting’s higher grades designate higher degree of cerebral injury. To score neurological findings, a 5-point scale was used, with no symptoms of neurological deficit=0, failure to fully extend left paw, 1; circling to left, 2; falling to left, 3; no walking ability spontaneously; and depressed consciousness, 4.

Infarct volume determination

After anesthetizing six rats per group, their brain tissues were isolated and immediately frozen at −20 °C. The samples were sliced into two mm-thick coronal cuts and underwent staining with 2 % (w/v) 2,3,5-triphenyl tetrazolium chloride (Sigma-Aldrich, USA) for 30 min at 37 °C before being immersed overnight in 4 % (w/v) paraformaldehyde. Image J software (National Institutes of Health, Bethesda, USA) was used to photograph and analyze the slices. Each slice’s white zones represented infarcted areas. The brain infarct volumes were calculated in percentages and normalized to each brain volume.

Brain water content estimation

After isolating the brains of the rats under deep anesthesia, the contralateral cortex of their brains was separated and the weight of wet tissue was measured, and then the samples were put in an oven to dry for 48 h at a temperature of 105 °C, and the dry weight of the tissue was also obtained. To calculate the brain water content, the difference in the weights of dry and wet tissue was calculated and divided by the wet weight, and the resulting value was multiplied by 100 to obtain the percentage of water content changes.

Quantification of indices of mitochondrial function

First, the mitochondrial content of the samples was isolated. For this purpose, peripheral zones of penumbra of ipsilateral cortex of the samples were placed in a solution containing isolating buffer plus a protease inhibitor cocktail (Sigma Aldrich, USA) to homogenize. Then the samples were centrifuged at 800 g and the resulting pellet was again centrifuged at 12,100 g. The BCA kit (Beyotime, Jiangsu, China) was also used to quantify the total protein of the mitochondrial fractions. In order to determine the levels mitochondrial ROS, the mitochondrial fraction was placed in a phosphate buffer solution containing 2 μmol DCFDA dye (Sigma-Aldrich, USA) at 37 °C for 30 min. Then, the absorbance of the solution was read at the excitation and emission wavelengths of 480 and 530 nm by a fluorometer. The absorbance values were normalized for the total protein of samples. Additionally, to estimate the mitochondrial membrane potential changes, the mitochondrial supernatant (100 μL) was added into a phosphate buffer solution containing 2 μL JC-1 dye- (Sigma-Aldrich, USA) and kept for 30 min at 37 °C. Using a fluorometer device, the red and green fluorescence intensities were obtained. The red to green intensity ratio was calculated to estimate the potential changes. Finally, the levels of manganese superoxide dismutase (MnSOD) were determined from the mitochondrial supernatant using the specific commercial ELISA kit (Cat. no: MBS1600375; MyBioSource, San Diego, California, USA), following the manufacturer’s instructions. The samples’ absorbance was measured at 450 nm, and the values were adjusted and presented as U/mg total protein content of mitochondrial samples determined using the BCA kit (Beyotime, Jiangsu, China).

Cytokines assays

Following 24 h of reperfusion, the ipsilateral cortex sample was homogenized in a lysis buffer (Beyotime, Jiangsu, China) and centrifuged at 8,000 g. Following the collection of sample supernatants, the levels of proinflammatory cytokines TNF-α (Cat. no: MBS175904), IL-1β (Cat. no: MBS825017), and IL-6 (Cat. no: MBS2021530) were measured by means of specialized ELISA kits according to manufacturer instructions (MyBioSource, San Diego, CA, USA). The cytokine levels of each sample were normalized by its protein contents (measured by the Bradford’s method), and the data were given as mg of protein per sample.

Immunoblotting

Proteins extracted from the samples underwent separation through polyacrylamide gel electrophoresis and subsequently transferred onto a polyvinylidene difluoride (PVDF) membrane. After washing steps and blocking the membranes in skim milk, they were treated with rabbit polyclonal antibodies against PGC-1α, NRF-1, and TFAM (1:1,000, Santa Cruz, USA) as well as beta-actin (1:2,000, Santa Cruz, USA). An enhanced chemiluminescence detection technique (Keygen Biotech, Nanjing, China) was employed for the visualization of the individual antibody–antigen responses. Quantity One 1-D Analysis Software (Bio-Rad, USA) was used to compute the target protein bands’ intensities, which were then adjusted to beta-actin intensity.

Statistical analysis

The data was presented as mean ± SD The one-way analysis of variance and Tukey post hoc test were used to analyze differences between groups. The p-value less than 0.05 was set to determine the minimal level of statistical significance.

The neuroprotective action of LIM

After induction of cerebral I–R injury by MCAO in rats, their neurological deficits were scored using a 5-point scale. In comparison to the Sham group, MCAO-receiving rats showed a severe neurological deficit, demonstrating the success of induction of MCAO injury model. When compared to the IR group, LIM-treated rats had less neurological deficit 24 h after ischemia, but the higher dose of LIM had a superior impact (p<0.001) than the lower dose (p<0.05) (Figure 1A). Additionally, compared to IR rats receiving LIM 100 mg/kg, blockage of mK–ATP channels via 5HD dramatically eliminated the LIM’s protective impact on neurological results (p<0.01). Moreover, compared to the IR group, 100 mg/kg LIM significantly lowered the brain water content (p<0.05) (Figure 1B). Cerebral infarct volumes were also measured by the tetrazolium chloride staining method to validate the neuroprotective benefits of LIM. The results revealed that LIM treatment at both dosages significantly and dose-dependently decreased IR-induced cerebral infarct sizes (Figure 1C and D). Inhibiting mK–ATP channels considerably reduced the effects of 100 mg/kg LIM on cerebral water content (p<0.05) and infarct volumes (p<0.01).

Figure 1:

Effect of LIM treatment on neuroprotective endpoints. (A) Grading of neurological deficits; (B) water contents of brain; (C) cerebral infarction volumes; (D) cerebral infarction images (n=6). **p<0.01, and ***p<0.001 vs. Sham group; #p<0.05, and ###p<0.001 vs. IR group; $p<0.05, and $$p<0.01 vs. IR+LIM100 group. IR, ischemia–reperfusion; LIM, limonene; 5HD, 5-hydroxydecanoate.

Effect of LIM on the mitochondrial activity

The measurement of mitochondrial ROS levels and mitochondrial membrane potential changes enabled the estimation of mitochondrial activity in this study. When compared to the Sham group, cerebral I–R damage significantly increased mitochondrial ROS production and depolarized mitochondrial membrane potential (p<0.001) (Figure 2A and B). But then again, LIM treatment in rats before reperfusion considerably and dose dependently reversed alterations in mitochondrial parameters induced by I–R injury (p<0.05 and p<0.001). Additionally, simultaneous administration of 5HD effectively eliminated the positive effects of LIM on the alterations in mitochondrial ROS and membrane potential (p<0.01) (Figure 2).

Figure 2:

Effect of LIM treatment on the levels of brain mitochondrial endpoints and proinflammatory cytokines production. (A) Mitochondrial ROS; (B) mitochondrial membrane potential; (C) mitochondrial MnSOD; (D) TNF-α; (E) IL-6; (F) IL-1β (n=6). ***p<0.001 vs. Sham group; #p<0.05, and ###p<0.001 vs. IR group; $p<0.05, $$p<0.01, and $$$p<0.001 vs. IR+LIM100 group. IR, ischemia–reperfusion; LIM, limonene; 5HD, 5-hydroxydecanoate.

Effect of LIM on the MnSOD levels

MnSOD, acting endogenously as a powerful antioxidant enzyme, was found in considerably lower levels in the mitochondrial fraction of cerebral tissue of the IR group than the Sham group (p<0.001) (Figure 2C). LIM substantially increased the IR-induced decrease of MnSOD at 100 mg/kg but not at 10 mg/kg (p<0.001). In contrast to the IR+LIM100 group, the administration of 5HD to block the opening of mK–ATP channel considerably reduced the LIM’s ability to increase MnSOD levels (p<0.05).

Effect of LIM on the proinflammatory cytokines

Cerebral tissue TNF-α (Figure 2D), IL-1β (Figure 2E), and IL-6 (Figure 2F) production was considerably increased when cerebral I–R damage was induced in rats as compared to that in the Sham group (p<0.001) (Figure 2). TNF-α levels were unaffected by LIM at 10 mg/kg; however, this dose significantly decreased IL-6 and IL-1β levels as compared to the IR group (p<0.05) (Figure 2D–F). In brain samples of IR rats treated with 100 mg/kg LIM, all proinflammatory cytokines were shown to be significantly downregulated compared to the IR group (p<0.01). Finally, blockage of mK–ATP channels substantially abolished anti-inflammatory effects of LIM 100 mg/kg (p<0.05).

Effect of LIM on the mitochondrial biogenesis proteins

PGC-1α, TFAM, and NRF1, three important proteins that regulate mitochondrial biogenesis, were dramatically downregulated after in vivo cerebral I–R damage in rats (p<0.001) (Figure 3A–C). LIM administration at a dose of 10 mg/kg was unable to reverse the decreased protein expression following I–R damage. In contrast to the IR group, the expression levels of PGC-1α (p<0.001), NRF1, and TFAM (p<0.01) were considerably increased following LIM treatment at 100 mg/kg. When compared to the IR+LIM100 group, 5HD’s blockade of mK–ATP channels significantly reduced the impact of higher doses of LIM on the expression of the biogenesis proteins (p<0.05) (Figure 3).

Figure 3:

Effect of LIM treatment on mitochondrial biogenesis proteins expression. (A) Representative immunoblots; (B) PGC-1α; (C) NRF1; (D) TFAM (n=4). ***p<0.001 vs. Sham group; ##p<0.01, and ###p<0.001 vs. IR group; $p<0.05 vs. IR+LIM100 group. IR, ischemia–reperfusion; LIM, limonene; 5HD, 5-hydroxydecanoate.