Introduction

Stroke, also known as cerebrovascular accident (CVA), is caused by rapid interruption of blood supply to the brain by the major cerebral arteries leading to impairment of central nervous system (CNS) functions (Deb et al. 2010). Stroke is the primary cause of disability because of functional impairments in the patients, and it is also a predisposing factor for epilepsy and depression. It has been reported that in India, 0.6 million people died due to stroke in the year 2000 and it is expected to increase to 0.95 million in 2020 (Ezzati et al. 2003). Out of total stroke population, 80% are ischemic stroke, and it is caused by the blockade of blood flow to the brain by the formation of embolism or thrombosis in cerebral blood vessels. Vascular occlusion results in a shortage of oxygen and energy, followed by the formation of reactive oxygen species, the release of glutamate, accumulation of intracellular calcium, and induction of inflammatory processes (Lipton 1999). This sequence of events called the ischemic cascade leads to irreversible tissue injury. The ischemic penumbra is an area of ischemic brain tissue that surrounds the infarcted core which is potentially restorable if appropriate treatment is administered within a specified therapeutic window (Fisher 2004).

Two major approaches are being developed to treat an ischemic stroke which is neuroprotection and reperfusion. The only accepted medical treatment for acute ischemic stroke is intravenous thrombolysis with rtPA (Tsai et al. 2015). The reperfusion therapy leads to an imbalance in the production of harmful reactive oxygen species (ROS) over endogenous anti-oxidant protection strategies (Shirley et al. 2014). In this situation, antioxidant drugs would be useful to tackle the post-reperfusion effect. Hence, a drug having both activities (antithrombotic and antioxidant) can be a better option for stroke treatment. Indole-3-carbinol (I3C), found in cruciferous vegetables, is a naturally occurring compound (Aggarwal and Shishodia 2006). It has been shown to have both antioxidant (Aggarwal and Shishodia 2006; El-Naga et al. 2014) and antithrombotic activities (Park et al. 2008) in a variety of animal models. I3C shows in vitro antiplatelet effect through the inhibition of GP IIb/IIIa receptor and thromboxane B2 formation (Park et al. 2008). Further, I3C decreased fatality of mice due to thromboembolism induced by collagen and epinephrine (Park et al. 2008). Furthermore, I3C was found to be effective against ischemic reperfusion injury of striated muscles by reducing the expression of leukocytic and endothelial adhesion proteins (Ampofo et al. 2017). Antiplatelet drugs have been used in the treatment of ischemic stroke (Sandercock et al. 1999). Hence, we presumed that I3C treatment may inhibit platelet aggregation and therefore may be useful in the treatment of cerebral ischemia.

I3C has a short half-life, rapid plasma clearance, and limited brain penetration (Anderton et al. 2004). However, oral dose of 50 mg/kg daily for 14 days was found to be effective against clonidine-induced depression in the rat (El-Naga et al. 2014). Further, I3C is biotransformed into active metabolite 3,3′-diindolylmethane (DIM). DIM also has been reported to protect neuronal cells by inhibiting lipopolysaccharide-induced microglial hyperactivation and attenuates neuroinflammation (Kim et al. 2013). Therefore, the neuroprotective effect could be due to both I3C and its metabolite DIM.

In view of the above facts, we investigated the pharmacological effect of I3C for the treatment of cerebral ischemic stroke in rat model. I3C and clopidogrel were given once daily through oral route after 2 h of ischemia or at the time of reperfusion and continued up to the 14th day. Ischemic stroke was induced by middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion. Neurological assessment was done by postural reflex, forelimb placing, and cylinder tests. To assess the recovery from cerebral ischemia, mean cortical blood flow and infarct volume were measured. The probable mechanism of action was evaluated through antioxidant activity and ADP-induced platelet aggregation ex vivo. Effect of I3C on ADP-induced platelet aggregation in human platelet-rich plasma (PRP) was done to determine its therapeutic potential. Furthermore, the in vivo antithrombotic activity of I3C (both pre-treatment and post-treatment) was evaluated in the kappa-carrageenan induced thrombosis rat model. In all experimental models of the present study, clopidogrel served as a positive control.

Materials and methods

Animals

Male Wistar rats (250–300 g) from the central animal house of the Institute of Medical Sciences, Banaras Hindu University, Varanasi, India, were used. Rats were housed under standard laboratory conditions, like free access to food and water, standardized number of animals per cage, timely exchange of bedding, and constant light and dark cycle. An acclimatization of 2 weeks was allowed before experiments began. Approval of Institutional Animal Ethics Committee (Protocol no. Dean/2016/CAEC/31) was granted before the beginning of the experiment. All the experiments were performed as per the guidelines of laboratory animal care (National Research Council US Committee for the Update of the Guide for the Care and Use of Laboratory Animals 2011).

Materials

I3C and pentobarbitone (Sigma-Aldrich, St.Louis, MO, USA); clopidogrel was donated by Ranbaxy Research Laboratories, Gurgaon, India; ADP (Hi-Media, Mumbai, India).

Preparation of solid dispersion of I3C

I3C was practically insoluble in water at the required dose of 50 mg/kg. The solid dispersion is one of the most widely used method for the oral administration of poorly soluble drug (Khodaverdi et al. 2012). Therefore, in the present study, the solid dispersion of I3C was prepared. It is prepared by solvent evaporation technique. I3C and polyethylene glycol (MW 5000) in the ratio of 1:9 were weighed and triturated in the mortar-pestle, the mixture was dissolved in methanol followed by evaporation of the solvent using rotavapor. The above mixture was dried for 12 h at 37 °C and was then dispersed in water according to the required doses of 12.5, 25, and 50 mg/kg (Singh et al. 2002). Fourier transform-infrared (FTIR) spectrometer of the prepared mixture was performed to determine drug and polymer interaction (Singh et al. 2002). Clopidogrel was suspended in 9% PEG 5000. To control and sham groups, vehicle (aqueous 9% PEG 5000) was administered.

Drug treatment

The study was conducted using two sets of experiments. The first set of experiment was performed to evaluate the treatment potential of I3C against cerebral ischemia in MCAO rat model. The second set of the experiment was performed for evaluating the antithrombotic activity of I3C in carrageenan-induced thrombosis rat model.

In MCAO experiment, male Wistar rats were separated into six groups (n = 11). They are control, Sham (vehicle), I3C (12.5 mg/kg), I3C (25 mg/kg), I3C (50 mg/kg) (El-Naga et al. 2014; Liu and McCullough 2011) and clopidogrel (15 mg/kg) (Yang et al. 2015). I3C and clopidogrel were given orally after 2 h of ischemia or at the time of reperfusion by oral gavage. The treatment was continued up to 14 days once daily as seen in Figs. 1 and 2. Animals were subjected to behavioral tests (n = 11) after 1 h, days 1, 7, and 14 to understand the improvement in neurological behavior. Blood samples were collected after 1 h of the last dose (Muddana et al. 2014; Muddana et al. 2015) for the ex vivo platelet aggregation (n = 6) study. Brain samples were taken out (Kumar et al. 2018; Shakya et al. 2011) for infarct volume (n = 4) and antioxidant (n = 4) studies.

Fig. 1
figure 1

FTIR spectra of pure I3C and I3C-PEG solid dispersion

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Fig. 2
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The experimental design using MCAO model

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In antithrombotic experiment, male Wistar rats were divided into five groups (n = 5) that is control given vehicle, clopidogrel (15 mg/kg), I3C (12.5 mg/kg), I3C (25 mg/kg), and I3C (50 mg/kg). The tail of the rat was tied with the surgical thread and immediately administered carrageenan (0.9 mg/kg, iv) to induce tail thrombosis. After 15 min of carrageenan administration, the thread was loosened. In pretreatment experiment, single dose of I3C and clopidogrel were administered before 60 min of tail ligation and in post-treatment experiment at the time of reperfusion (thread removal). The length of the infarcted tail region was measured using laser speckle Doppler imager (Omegazone OZ-2 STD, Japan) at 0, 2, 4, 8, 12, 24, and 36 h post-reperfusion.

Middle cerebral artery occlusion in rat

The intraluminal suture occlusion method (Longa et al. 1989) was used to occlude right middle cerebral artery for 2 h with modification (Belayev et al. 1996). In this, a nylon monofilament of number 3.0 was inserted retrogradely into MCA. In this technique, the right common carotid artery (CCA) with its bifurcation into external carotid artery (ECA) and internal carotid artery (ICA) was exposed. Then the occipital branches of ECA were ligated, and the suture was introduced in ECA proximal to the bifurcation point. From the bifurcation point, 18–20 mm filament was passed via ECA to MCA. Prior to use of filament in arteries, it was dipped in poly-lysine solution so as to enhance its linkage to endothelium and enhance the reproducibility of infarct result. The incision in neck region was sutured. After 2 h of MCAO, animals were re-anesthetized, and the filament was carefully removed. Animals were returned to cages after stitches and betadine was applied over the incision. Further meloxicam as an analgesic at the dose of 0.3 mg/kg was administered to all the animal before and 24 h after the surgery. The body temperature of the surgery animal was maintained at 36.5 °C ± 0.5 °C using a temperature-controlled heating plate till 2 h of the reperfusion. Cerebral blood flow was measured to confirm blood vessel occlusion using a laser speckle blood flow imaging system (Omegazone OZ-2 STD, Japan).

Platelet aggregation study

Preparation of platelets

Human blood was withdrawn from healthy volunteer for in vitro study after obtaining their informed consent. Whole rat blood was collected from the rat on day 14 after 1 h of the last dose through cardiac puncture for ex vivo study. The blood was collected, into plastic syringes containing 1000 U sodium heparin/ml (in 0.9% saline) solution. Platelet-rich plasma (PRP) from rat blood and human blood was collected by polyethylene pipette into a plastic tube after centrifugation at 1200 rpm for 5 min and 1600 rpm for 10 min at 4 °C, respectively. The residual blood was centrifuged again as above and PRP was also collected. These procedures were repeated three times. Platelet-poor plasma (PPP) from rat and human blood were prepared by centrifugation of the residual blood at 4000 rpm for 5 min and 2700 rpm for 10 min at 4 °C, respectively (Dhurat and Sukesh 2014).

Platelet aggregation study

The inhibitory effect of I3C treatment (in vitro in human PRP and ex vivo in rat PRP) was determined by the optical method, using a dual channel Aggregometer (Chrono-Log Co., Havertown, PA, USA). The baseline value was set using PRP and maximal transmission using PPP. PRP of different group was collected and stirred at 1200 rpm. Then, 10 μM ADP was added to the platelets. Changes in light transmission were recorded for 5 min after stimulation (Born and Wehmeier 1979).

Behavioral parameters

Standardized battery of tests was used to measure sensorimotor neurological function in all animals after 1 h, 24 h, 7th, and 14th day of treatment (Belayev et al. 1996). The tests consisted of two separate tests, i.e., postural reflex test and forelimb placing test. Upper body posture was inspected by postural reflex test while the rat was hanged by the tail (Bederson et al. 1986). Sensorimotor integration was examined by forelimb placing test using visual, tactile, and proprioceptive stimuli (De Ryck et al. 1989). Further, the locomotor asymmetry of forelimb was examined by cylinder test (Hua et al. 2002). Higher value indicates more severe motor deficits and vice versa. All the experiments were performed by the scholar blinded to the treatment groups. Neurological function was evaluated on a scale of 0 to 12 (normal score = 0, maximal score = 12) as previously explained (Belayev et al. 1996).

Estimation of mitochondrial oxidative stress

Lipid peroxidation (LPO) or malondialdehyde (MDA) estimation

Malondialdehyde, a measure of membrane lipid peroxidation, was quantified by reaction with thiobarbituric acid reactive substances (TBARS) at 532 nm by the method of Ohkawa et al. (1979). The chromophore MDA-TBARS formed in the reaction was extracted into an organic layer, and the absorbance was measured at 532 nm. The results were expressed as nmol MDA/mg protein.

Superoxide dismutase estimation

Superoxide dismutase (SOD) activity was assayed according to the method of Kakkar (1984). Blue colored formazan was formed and measured at 560 nm (Kakkar et al. 1984).

Catalase estimation

Catalase activity was assayed following the method of Luck (1974). The UV absorption of hydrogen peroxide was calculated at 240 nm, whose absorbance decreases when degraded by the enzyme catalase. From the decrease in absorbance, the enzyme activity was calculated.

Infarct volume measurement

The rats were euthanized and brain tissue was collected on day 14. Rat brains of each group were frozen at − 20 °C for 5 min and sectioned into 2 mm-thick coronal slices. Slices were incubated for 20 min at 37 °C in 2% triphenyl tetrazolium chloride (TTC) solution in phosphate-buffered saline (PBS, pH 7.4). Color images of these slices were captured and size of infarction was calculated using ImageJ software. To obtain the total infarct area, infarcted areas of all sections were added. Further, brain edema may impair the exact infarction volume and thus the obtained infarct area was corrected. The corrected infarct volume was calculated as follows: corrected infarct area = measured infarct area * {1-[(ipsilateral hemisphere area-contralateral hemisphere area)/contralateral hemisphere]}. To determine the infarct volume, total infarct area was multiplied by the thickness of the brain sections (Berti et al. 2002; Paliwal et al. 2017).

Cerebral blood flow (CBF) recording

Changes in CBF were recorded by a laser speckle blood flow imaging system (omegazone OZ-2 STD). Skull of anesthetized rats were exposed by a midline scalp incision and placed on the black sponge sheet that located under the arm stand. Arm stand holds the CCD camera, the lens (ZM10-18, MF12), and the laser unit (780 nm for measurement and 650 nm for positioning). Raw speckle images were recorded from the skull surface using LSI Software (LSI ver.3.3, Omegawave, Inc., Tokyo, Japan) and average cerebral blood flow was determined by further analysis of images by using LIA Software (LIA ver.3.3, Omegawave, Inc., Tokyo, Japan). The black sheet does not reflect the laser light and the effect makes the blood flow image clear (Atlan et al. 2007; Guo et al. 2010; Krishnamurthy et al. 2015).

Carrageenan-induced rat tail thrombosis

The in vivo antithrombotic activity of I3C was evaluated in the carrageenan induced thrombosis rat model (Hagimori et al. 2009). Twenty-five male rats were separated into five groups: vehicle (aqueous 9% PEG 5000), clopidogrel (15 mg/kg), I3C (12.5 mg/kg), I3C (25 mg/kg), and I3C (50 mg/kg), each group containing five rats. After 60 min of oral dosing (vehicle, I3C, and clopidogrel), the tail of the animal was ligated followed by administration of carrageenan (0.9 mg/kg) through intravenous injection. Ligatures were removed after 15 min of injection. The length of the blocked tail, showing segment of the tail being devoid of blood, was measured using laser speckle blood flow imaging system at 0, 2, 4, 8, 12, 24, and 36 h post-reperfusion. Further, a separate set of experiment was done to study the post-treatment effect of I3C. I3C (12.5, 25, and 50 mg/kg) was administered at the time of reperfusion (ligature removal) and the same procedure as for the pretreatment study was followed thereafter.

Statistical analysis

Repeated measures of two-way ANOVA was performed for the analysis of behavioral, antithrombotic, and CBF data followed by Bonferroni’s post hoc test. For platelet aggregation and biochemical data analysis, one-way ANOVA was performed followed by Student Newman Keuls post hoc test. A level of p < 0.05 was considered as significant in all the data analysis.

Results

FTIR spectra

The FTIR studies showed no change in the spectral characteristics of drug both pure and in solid dispersion form as seen in Fig. 1.

Effect of I3C on platelet aggregation

I3C potently inhibited ADP-induced human platelet aggregation in vitro in a concentration-dependent manner as shown in Fig. 3A. Statistical analysis using one-way ANOVA revealed significant differences among the groups [F (5, 30) = 128.7: P < 0.05]. The post hoc analysis revealed, I3C dose dependently inhibited ADP-induced platelet aggregation in human PRP compared to vehicle. Figure 3B illustrates the effect of oral I3C treatment (12.5, 25, and 50 mg/kg) on ADP-induced platelet aggregation in rat PRP. One-way ANOVA followed by post hoc analysis revealed, I3C at all three doses significantly inhibited ADP-induced platelet aggregation in rat PRP compared to vehicle [F (4, 25) = 95.16: P < 0.05].

Fig. 3
figure 3

The effect of I3C on ADP-induced platelet aggregation in human PRP (in vitro) and rat PRP (ex vivo). A The effect of I3C (3, 6, 12 and 25 μM) on ADP-induced platelet aggregation in healthy human PRP. Platelet aggregation was induced by adding 10 μM ADP and terminated after 5 min, and monitored by measuring light transmission with an aggregometer. Data show the mean ± SD (n = 6). aP < 0.05 compared to vehicle group, bP < 0.05 compared to I3C 3 μM, and cP < 0.05 compared to I3C 6 μM [one-way ANOVA followed by Student Newman Keuls post hoc test]. B Ex vivo effects of I3C on ADP induced platelet aggregation after 2-h intraluminal suture occlusion of MCA and 14 days reperfusion. I3C was orally administered to male rats daily for 14 days to three test groups at the doses of 12.5 mg/kg, 25 mg/kg, and 50 mg/kg, and clopidogrel served as positive control group at the dose of 15 mg/kg. Blood was collected 1 h after the final treatment. Data show the mean ± SD of six rats. aP < 0.05 compared to vehicle group, bP < 0.05 compared to I3C12.5, and cP < 0.05 compared to I3C25 [one-way ANOVA followed by Student Newman Keuls post hoc test]

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Effect of I3C on mean CBF

The impact of I3C (12.5, 25, and 50 mg/kg) on changes in mean CBF in MCAO rats is depicted in Fig. 4. Statistical analysis using two-way ANOVA showed the significant differences in mean CBF among groups [F (4, 175) = 54.42; P < 0.05], time [F (6, 175) = 990.7; P < 0.05], and interaction between groups and time [F (24, 175) = 11.11; P < 0.05]. Post hoc test confirmed that I3C significantly increased in mean CBF after 4 and 6 h of treatment.

Fig. 4
figure 4

The effect of I3C (12.5, 25, and 50 mg/kg) on changes in mean CBF in MCAO rats. All values are Mean ± SD (n = 6). aP < 0.05 compared to vehicle [repeated measures of two-way ANOVA followed by Bonferroni post hoc test]

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Effect of I3C on MCAO induced changes in behavior

Postural reflex test

Figure 5A shows the effect of I3C (12.5, 25, and 50 mg/kg) and clopidogrel (15 mg/kg) on changes in average score in the postural reflex test on MCAO rats. Significant differences in average score among groups [F (4, 200) = 84.21; P < 0.05], time [F (3, 200) = 181.9; P < 0.05], and interaction between groups and time [F (12, 200) = 10.16; P < 0.05] was found using two-way ANOVA statistical analysis. Post hoc analysis revealed that at 1 h of the experimental protocol, there were no significant differences in average score among the groups. However, on day 1, I3C (25 and 50 mg/kg) and clopidogrel exhibited significant reduction in average score compared to vehicle group; this effect was retained up to day 14. The I3C (12.5 mg/kg) dose showed significant reduction in average score compared to vehicle on 7th day and the effect was maintained up to day 14.

Fig. 5
figure 5

The effect of I3C (12.5, 25 and 50 mg/kg) and clopidogrel (15 mg/kg) on changes in behavioral test in stroke rats after 2-h intraluminal suture occlusion of MCA and 14 days reperfusion. A shows changes in average score in the postural reflex test in stroke rats. B shows changes in forward visual limb placing score in the forelimb placing test. C shows changes in lateral visual limb placing score in the forelimb placing test. D reveals changes in tactile forward limb placing score in the forelimb placing test. E shows changes in tactile lateral limb placing score in the forelimb placing test. F shows changes in proprioceptive limb placing score in the forelimb placing test on MCAO rats. All values are Mean ± SD (n = 11). aP < 0.05 compared to vehicle, bP < 0.05 compared to I3C12.5, cP < 0.05 compared to I3C25, xp < 0.05 compared to 1 h and yp < 0.05 compared to 1 day [Repeated measures of two-way ANOVA followed by Bonferroni post hoc test]

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Forward visual limb placing

The impact of I3C (12.5, 25 and 50 mg/kg) and clopidogrel (15 mg/kg) on alteration in average score in the forward visual limb placing test of MCAO rats is demonstrated in Fig. 5B. Significant differences in average score among groups [F (4, 200) = 118.2; P < 0.05], time [F (3, 200) = 297.1; P < 0.05], and a statistically considerable interaction between groups and time [F (12, 200) = 13.71; P < 0.05] was obtained using two-way ANOVA. On day 1, all three I3C doses and clopidogrel showed a significant reduction in average score compared to vehicle group; this effect persisted up to day 14.

Lateral visual limb placing

Figure 5C shows the effect of I3C (12.5, 25, and 50 mg/kg) and clopidogrel (15 mg/kg) on changes in lateral visual limb placing score in the forelimb placing test on MCAO rats. Statistical analysis by repeated measure two-way ANOVA revealed significant differences in average score among groups [F (4, 200) = 67.80; P < 0.05], time [F (3, 200) = 206.4; P < 0.05], and interaction between groups and time [F (12, 200) = 8.05; P < 0.05]. Post hoc analysis revealed that I3C and clopidogrel showed significant reduction in average score compared to vehicle group from day 1 onwards which was retained up to day 14.

Tactile forward limb placing

The effect of I3C (12.5, 25, and 50 mg/kg) and clopidogrel (15 mg/kg) on changes in tactile forward limb placing score in the forelimb placing test on MCAO rats is depicted in Fig. 5D. Significant differences in average score among groups [F (4, 200) = 95.68; P < 0.05], time [F (3, 200) = 276.2; P < 0.05], and a statistically considerable interaction between groups and time [F (12, 200) = 9.25; P < 0.05] was observed by two-way ANOVA. Treatment with 13C and clopidogrel showed significantly improvement in the tactile forward limb placing response from day 1 onwards.

Tactile lateral limb placing

Figure 5E shows the effect of I3C (12.5, 25, and 50 mg/kg) and clopidogrel (15 mg/kg) on changes in tactile lateral limb placing score in the forelimb placing test on MCAO rats. Statistical analysis revealed significant differences in average score among groups [F (4, 200) = 153.3; P < 0.05], time [F (3, 200) = 302.6; P < 0.05], and interaction between groups and time [F (12, 200) = 15.70; P < 0.05]. On day 1, I3C in all three doses and clopidogrel showed a significant reduction in average score compared to vehicle group; this effect was retained up to day 14.

Proprioceptive limb placing

The effect of I3C (12.5, 25, and 50 mg/kg) and clopidogrel (15 mg/kg) on changes in proprioceptive limb placing score in the forelimb placing test on MCAO rats is depicted in Fig. 5F. Significant differences in average score among groups [F (4, 200) = 166.9; P < 0.05], time [F (3, 200) = 272.5; P < 0.05], and a statistically considerable interaction between groups and time [F (12, 200) = 13.97; P < 0.05] was found using two-way ANOVA statistical analysis. Post hoc analysis revealed that at 1 h of the experimental protocol, there were no significant differences in average score among the groups. However, on day 1, I3C (25 and 50 mg/kg) and clopidogrel exhibited significant reduction in average score compared to vehicle group. The I3C (12.5 mg/kg) dose showed significant reduction in average score compared to vehicle on 7th day; this effect was retained up to day 14.

Total neurological score

Figure 6 shows the effect of I3C (12.5, 25, and 50 mg/kg) and clopidogrel (15 mg/kg) on the total neurological score on MCAO rats. Statistical analysis by repeated measure two-way ANOVA revealed significant differences in average score among groups [F (4, 200) = 132.3; P < 0.05], time [F (3, 200) = 359.2; P < 0.05], and a statistically significant interaction between groups and time [F (12, 200) = 11.85; P < 0.05]. Drug-treated rats showed a significant reduction in average score compared to rats given vehicle from day 1 onwards; this effect persisted up to day 14.

Fig. 6
figure 6

The effect of I3C (12.5, 25 and 50 mg/kg) and clopidogrel (15 mg/kg) on the total neurological score after 2-h MCAO and 14 days reperfusion. All values are Mean ± SD (n = 11). aP < 0.05 compared to vehicle administered, bP < 0.05 compared to I3C12.5, cP < 0.05 compared to I3C25, xp < 0.05 compared to 1 h and yp < 0.05 compared to 1 day [repeated measures of two-way ANOVA followed by Bonferroni post hoc test]

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Cylinder test

The effect of I3C (12.5, 25, and 50 mg/kg) and clopidogrel (15 mg/kg) on changes in asymmetry score in the cylinder test in MCAO rats is represented in Fig. 7. Statistical differences in asymmetry score among groups [F (4, 200) = 314.9; P < 0.05], time [F (3, 200) = 541.7; P < 0.05], and interaction between groups and time [F (12, 200) = 35.38; P < 0.05] were observed. Post hoc analysis showed that treated rat exhibited a significant reduction in asymmetry score compared to vehicle administered rats from day 1 onwards; this effect persisted up to day 14.

Fig. 7
figure 7

The effect of I3C (12.5, 25 and 50 mg/kg) and clopidogrel (15 mg/kg) on changes in asymmetry score in the cylinder test after 2-h MCAO followed by 14 days reperfusion. All values are Mean ± SD (n = 11). aP < 0.05 compared to vehicle administered, bP < 0.05 compared to I3C 12.5, cP < 0.05 compared to I3C 25, xp < 0.05 compared to 1 h and yp < 0.05 compared to 1 day [repeated measures of two-way ANOVA followed by Bonferroni post hoc test]

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Effect of I3C on mitochondrial oxidative stress

Lipid peroxidation level

Figure 8A shows the effect of I3C (12.5, 25, and 50 mg/kg) on LPO (MDA) level in brain of MCAO rats. Statistical analysis with one-way ANOVA showed significant differences among groups [F (5, 18) = 497.1: P < 0.05]. The post hoc analysis revealed that MCAO significantly increased the LPO level. All three doses of I3C significantly decreased the LPO level. The highest dose of I3C (50 mg/kg) significantly decreased the LPO level compared to other two doses.

Fig. 8
figure 8

The effect of I3C and clopidogrel on LPO, catalase and SOD level in the brain of MCAO rats after 2-h intraluminal suture occlusion of MCA and 14 days reperfusion. A shows the effect of I3C (12.5, 25, and 50 mg/kg) on LPO (MDA) level in the brain of MCAO rats (TBARS; Thiobarbituric acid reactive substances are formed as a byproduct of lipid peroxidation). B represents the effect of I3C (12.5, 25, and 50 mg/kg) on catalase level in the brain of MCAO rats. C illustrates the effect of I3C (12.5, 25, and 50 mg/kg) on SOD level in the brain of MCAO rats. All values are Mean ± SD (n = 4). aP < 0.05 compared to control group, bP < 0.05 compared to vehicle group, cP < 0.05 compared to I3C12.5, and dP < 0.05 compared to I3C25 [one-way ANOVA followed by Student Newman Keuls post hoc test]

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Catalase level

The effect of I3C treatment on catalase level in brain of MCAO rats is shown in Fig. 8B. Significant differences was obtained among groups [F (5, 18) = 351.2: P < 0.05] using one-way ANOVA. MCAO significantly decreased the CAT level and treatment with I3C at all three doses significantly increased the CAT level. I3C (50 mg/kg) dose significantly increased the CAT level compared to other two doses.

Superoxide dismutase level

Figure 8C demonstrates the effect of I3C (12.5, 25, and 50 mg/kg) on SOD level in brain of MCAO rats. One-way ANOVA result showed significant differences among groups [F (5, 18) = 38.83: P < 0.05]. Post hoc analysis showed that the level of SOD significantly decreased in MCAO group. All three doses of I3C significantly increased the SOD level.

Effect of I3C on infarction volume after MCAO

Figure 9 illustrates the effect of I3C formulation (12.5, 25, and 50 mg/kg) on infarct volumes in MCAO rats. One-way analysis of variance [F (5, 18) = 106.50; P < 0.05] followed by Bonferroni posttests revealed significantly smaller infarct volumes in I3C-treated group in contrast to the vehicle.

Fig. 9
figure 9

Quantitative analysis of the infarct volume at 14 day following reperfusion. I3C (12.5, 25, and 50 mg/kg) and clopidogrel (15 mg/kg) significantly reduced infarct volume from 29 ± 2.4% (180 ± 15 mm3) caused by middle cerebral artery occlusion to 21 ± 2.6, 14 ± 2.4, 9 ± 1.7 and 8 ± 1.3 respectively. All values are Mean ± SD (n = 4). aP < 0.05 compared to control group, bP < 0.05 compared to vehicle group, cP < 0.05 compared to I3C12.5, and dP < 0.05 compared to I3C25 [one-way ANOVA followed by Bonferroni post hoc test]

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Effect of I3C on thrombosis

I3C pretreatment potently suppressed carrageenan-induced rat tail thrombosis in a concentration-dependent manner as shown Fig. 10. Significant differences among groups [F (4, 175) = 865.6: P < 0.05], time [F (6, 175) = 437.00: P < 0.05], and interaction between group and time [F (24, 175) = 30.43: P < 0.05] were obtained using two-way ANOVA statistical analysis. The post hoc analysis revealed that treated groups showed a significant decrease in length of thrombus compared to vehicle group doses dependently. Whereas, post-treatment with I3C (12.5, 25, and 50 mg/kg) was ineffective against carrageenan-induced thrombus length (data not shown).

Fig. 10
figure 10

The effect of I3C (12.5, 25, and 50 mg/kg) and clopidogrel (15 mg/kg) pretreatment on carrageenan-induced rat tail thrombosis model. All values are Mean ± SD (n = 5). aP < 0.05 compared to vehicle group, bP < 0.05 compared to I3C12.5, cP < 0.05 compared to I3C25 and dP < 0.05 compared to I3C50 [two-way ANOVA followed by Bonferroni post hoc test]

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Discussion

We here report that I3C treatment alleviates the neurological deficits of cerebral ischemia along with reduced cerebral infarct volume and inhibits ADP-induced platelet aggregation. Further, in another experiment, I3C pretreatment inhibited carrageenan-induced rat tail thrombus length indicating prophylactic anti-thrombotic activity.

Cerebral ischemia is commonly linked with impaired sensorimotor function and cognitive function (Belayev et al. 1996). Due to the loss of limb function after stroke, behavioral test mostly focus on motor and sensory tests (Schaar et al. 2010). The neuro-behavioral test was performed for the assessment of the degree of damage over a period of time. The important focus of stroke treatment is the regain of behavioral function (Schaar et al. 2010). Administration of all three doses of I3C significantly improved the neurological scores as discussed below within 24 h of treatment. Ceiling effect of I3C was observed from day 7.

We first evaluated the postural reflux, which is used for evaluation of motor performance in cerebral ischemic rats (Bederson et al. 1986). Postural reflux score increased in MCAO rats and treatment with I3C in all three doses mitigated the reflux score. The limb placing score measures the hind and forelimb motor activity in neurobiological disorder and stroke rats (De et al. 1989). I3C treatment significantly decreased limb placing score increased in MCAO rats indicating improved motor performance.

Exploratory behavior of the rat is used to explore the neural basis of spatial and motor behavior. MCAO rats have shown asymmetry of forelimb usage in cylinder test which was consistent with an earlier study (Hua et al. 2002). Treatment with I3C attenuated the changes in asymmetric scoring in cylinder test and other neurobehavior changes in experimental rats. This suggests that I3C facilitates the spontaneous forelimb use after ischemic reperfusion injury.

Ischemic injury results due to the cessation of blood supply in the brain (Fahlenkamp et al. 2014; Weaver and Liu 2015). I3C improved cerebral blood flow which was reduced in cerebral ischemic rats. Studies have shown that restoring the blood supply in the brain alleviates neurological impairment in the rats (Belayev et al. 2003; Li et al. 2014; Yamauchi et al. 2017). There was sustained increase in CBF from 4 h after I3C administration in all the doses. A robust 60% of baseline increase in CBF was observed at 6 h with the lowest dose of I3C. Several studies have reported that use of antiplatelet drugs lead to recovery of blood flow after 6 h post reperfusion in ischemic model (Belayev et al. 2008; Yamauchi et al. 2017). Similarly, in our present study I3C inhibited platelet aggregation. This may be the reason for CBF enhancement, which occurs from 4 h after reperfusion with I3C treatment.

Oxidative stress induces lipid peroxidation and modifies the antioxidant defense system in the brain tissue under ischemic conditions (Thiyagarajan and Sharma 2004). The brain is vulnerable to oxidative stress due to the presence of non-heme iron in its environment, which is involved catalytically in the production of free radicals (Yousuf et al. 2005). The close connection among oxidative stress and cerebral ischemia has created substantial interest in the development of antioxidant therapies to treat ischemia-induced damage. The antioxidant potential of I3C may be responsible for observed effect by reducing the level of oxidative stress. I3C has been reported to shown neuroprotective effect against clonidine-induced neurotoxicity through antioxidant mechanism (El-Naga et al. 2014; Li et al. 2014). Lipids are the macromolecules most prone to oxidative stress, which plays a main role in the generation of MDA (Nazam Ansari et al. 2008). Earlier reports showed that MCA occlusion followed by reperfusion increased the MDA formation (Ban et al. 2012). Treatment with I3C significantly reduced the brain tissue MDA level in MCAO injury. This indicated that I3C was helpful in reducing the oxidative stress of MCAO. The existence of several antioxidant enzymes in the brain, including SOD, and CAT, protects the brain tissues from the damaging effect of oxidative stress caused by the formation of free radicals (Cui et al. 2004). SOD specifically reacts with the superoxide ion to form H2O2 while CAT detoxifies the so formed H2O2 into the water. Oxidative stress reduces the activity of these enzymes (Tripathi et al. 2017). In the present study I3C significantly increased the endogenous activity of SOD and CAT enzymes.

Further, oxidative stress resulting from focal cerebral ischemia leads to activation and accumulation of platelet in brain injured microvascular bed (Freedman 2008; Abumiya et al. 2000). The injured vascular wall releases ADP, platelet aggregation agonist (Choudhri et al. 1998) which enhances thrombus formation even after reperfusion of the MCA and is responsible for the post-ischemic hypoperfusion and ongoing neuronal damage (Choudhri et al. 1998). I3C inhibited ADP-induced platelet aggregation ex vivo. Similarly, SolCD39 (soluble form of Cluster of Differentiation 39) also known as ectonucleoside triphosphate diphosphohydrolase-1 inhibited ADP-induced platelet aggregation in vitro (Marcus et al. 1997) and in vivo (Pinsky et al. 2002), and improved the neurological score, postischemic cerebral perfusion and reduced total infarct volume (Belayev et al. 2003; Cha et al. 2008). Therefore, the observed recovery in neurological deficits and reduction in infarct volume with I3C treatment against MCAO-induced injury could be due to the inhibition of ADP-induced platelet aggregation. Additionally, I3C also inhibited ADP-induced platelet aggregation in vitro in human PRP. This indicates the clinical potential of I3C in conditions of cerebral ischemia.

Furthermore, we observed that I3C pretreatment shows antithrombotic action in vivo against carrageenan-induced rat tail thrombosis. I3C has been reported to decrease the mortality in mice caused due to pulmonary thrombosis evoked by collagen and epinephrine (Park et al. 2008). Therefore, I3C shows prophylactic anti-thrombotic activity.

In summary, our results show that I3C ameliorates MCAO-induced neurobehavioral deficits, increased cerebral blood flow, and reduced ischemic infarct volume. Anti-cerebral ischemic activity of I3C may be due to its ability to inhibit ADP-induced platelet aggregation and antioxidant activity. In addition, I3C shows prophylactic anti-thrombotic activity against carrageenan-induced thrombosis. Therefore, this preclinical study points to I3C as a potential compound for the treatment of cerebral ischemia.