Targeting the Proline-Rich Tyrosine Kinase 2 /Mitochondrial Calcium Uniporter Pathway, Ruthenium Red, and Nimodipine alone and in Combination Evoked Neuroprotective Effect in Experimentally Induced Brain Stroke in Rats
Yasmin M Etman2, Amany A Alzokaky1,3*, Wedad A Hassan b Ahmed H Eid2 and Azza S Awad1,4
1Pharmacology and Toxicology Department, Faculty of Pharmacy for Girls, Al-Azhar University, Egypt
2Pharmacology Department, National Organization for Drug Control and Research (NODCAR), Egypt
3Pharmacology and biochemistry Department, Faculty of Pharmacy, Horus University, Egypt
4Pharmacology and Toxicology Department, Faculty of Pharmacy, Ahram Canadian University, Egypt
Received Date: 23/06/2020; Published Date: 21/07/2020
*Corresponding author: Amany A Alzokaky, Lecturer in Pharmacology and Toxicology Department, Faculty of Pharmacy for Girls, Al-Azhar University, Cairo, Egypt
Cite this article: Amany A. Alzokaky, Targeting the proline-rich tyrosine kinase 2 /Mitochondrial calcium uniporter pathway, ruthenium red, and nimodipine alone and in combination evoked neuroprotective effect in experimentally induced brain stroke in rats, Op Acc J Bio Sci & Res 3(3)-2020.
Mitochondrial dysfunction caused by Ca2+ overload plays an essential role in ischemia associated with brain damage. The proline-rich tyrosine kinase 2 (Pyk2) /Mitochondrial calcium uniporter (MCU) pathway is essential in cerebral ischemic stroke and is responsible for mitochondrial damage and neuronal degeneration. The present study aimed to investigate the neuroprotective potentials of ruthenium red and nimodipine in brain ischemia induced by middle cerebral artery occlusion (MCAO). Ruthenium red (RR), nimodipine (NMD), and their combination effectively decreased the brain ischemic changes and mitigated the neuronal damage evidenced by reversing the brain histopathological aberrations and decreasing percentage of infarction volume. At the same time, both agents and their combination improved cerebral blood flow through the downregulation of Pyk2 and MCU gene expression in brain tissue. They also opposed the inflammatory load via attenuation of brain myeloperoxidase activity, lowered lipid peroxides, NAD(P)H dehydrogenase quinone 1(NQO1), and nitric oxide and boosted the brain glutathione. Moreover, they decreased the infraction volume. To the greatest of our knowledge, this is the first study to reveal the neuroprotective effects of RR, NMD, and their combination in the cerebral brain stroke model.
Keywords: Ruthenium red; Nimodipine; MCAO; Pyk2/MCU pathway; NQO1; Infraction volume.
A cerebral brain stroke is a universal brain injury. Stroke is a major cause of death in the world. An ischemic stroke caused by either a sudden reduction or complete blockage of the cerebral flow of the blood accounts for 85% of all cerebral stroke patients .There are several cerebral ischemia pathophysiology mechanisms, such as the discharge of excitatory neurotransmitters, an increase of oxidative stress, intracellular calcium levels, inflammation, and apoptosis .Oxidative stress, which plays an essential role in the pathogenesis of cerebral ischemia is caused by a difference between the manufacturing of reactive oxygen species (ROS) and its useful removal by endogenous scavenger enzymes and protective antioxidants [3,4] The mitochondria is the major participant in cell death and is specifically caught up in ischemia-induction of neuronal damage .
In brief, mitochondrial damage and morphological changes include
1. activation of the mitochondrial permeability transition pore (MPTP);
2. release of cytochrome C and apoptotic inducible factor;
3. decreased production of ATP and increased production of free radicals by dysfunction of the mitochondrial respiratory chain  and
4. Mitochondrial Ca2+overload, which activates proteases and finally ends with cell death. The regulatory mechanisms underlying the increase in the concentration of cytoplasmic Ca2+ after brain infarction-induced Ca2+overload are well understood and include the release of free radicals, activation of calcium-dependentkinases, and the start of apoptosis. These processes ultimately result in neuronal death.
Mitochondrial calcium uniporter (MCU) is a complex uniporter channel that mediates Ca2+ uptake into the mitochondrial matrix to regulate cell metabolism, cytoplasmic calcium signaling, and cell death  The knockdown of MCU has been proved to reduce mitochondrial Ca2+influx, protect mitochondrial function, and prevent neuronal death . Proline-rich tyrosine kinase 2 (Pyk2) is a 116-kDa cytoplasmic tyrosine kinase that is a member of the focal adhesion kinase family . Previous studies have confirmed that activation of Pyk2 in cerebral ischemia regulates N-methyl-d-aspartate-induced neuronal death [10,11]. However, further invivo experiments are required to better recognize the function of the MCU in neurodegeneration and the application of the MCU antagonist in neurodegenerative disorder therapy.
Ruthenium red affects inhibition of mitochondrial uniporter and selective intracellular ryanodine receptor (RyR) antagonist . Ruthenium red (RR) has been investigated to prevent the brain injury after cerebral ischemic stroke by inhibiting mitochondrial calcium uniporter which participates in ischemic damage  In past reports, ruthenium red has been studied to provide beneficial properties by declining brain inflammation,  and oxidative stress .
One of the well-known calcium channel blockers is the nimodipine (NMD). The human body naturally responds to hemorrhage by contracting the blood vessels to slow blood flow from the injured site. Conversely, stopping blood flow when the hemorrhage is in the brain sites causes more brain damage. NMD is thought to do its action by providing relaxation in the contracted blood vessels near the area of bleeding in cerebral hemorrhage so blood can flow more with no trouble. This effect reduces brain damage. NMD could be efficient in minimizing ischemia/reperfusion injury in the tissue of the ovary when exposed to ischemia .The purpose of the present study is to investigate the neuroprotective potentials of ruthenium red, nimodipine, and their combination in brain ischemia induced by middle cerebral artery occlusion (MCAO).
Materials and Methods
Animals and Surgical Preparation
All experimental procedures, in addition to animal handling, were carried out by the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication No. 85-23, revised 2011). The current protocol was approved by the Research Ethics Committee of Faculty of Pharmacy, Al-Azhar University for girls, Egypt (Number: PT 173, 2017). Adult male Sprague–Dawley rats weighing 200–270 g were used. Left cerebral artery occlusion was done to induce cerebral ischemia, as reported by Cummins& his co-workers . Chloral hydrate was used for anesthesia in a dose of 350 mg/kg, intraperitoneally . middle neck incision was done followed by left carotid artery occlusion using 0.5 cm stainless steel bulldog. The occlusion lasted for one hour and the skin was closed in this period for induction of ischemia then reperfusion was done for this artery for 24 hours. During the operation, the body temperature was kept at 37±0.5 °C with a heating lamp and blanket. The sham-operated rats were handled similarly, except the left cerebral artery was not occluded after the neck incision.
Ruthenium red as red crystals soluble in water and normal saline and nimodipine as white fine powder were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Nimodipine was dissolved in 50% dimethyl sulfoxide.
Design of the work
Sixty-four male rats were randomly divided into 8 groups (n=8): (1) sham group, (2) vehicle group, (3) MCAO group, (4) NMD group alone (10 mg/kg, i.p.), (5) NMD with MCAO group, (6) RR group alone (3 mg/kg, i.p.), (7) RR with MCAO group, (8) NMD and RR combination with MCAO group.
Determination of NAD(P)H dehydrogenase (quinone 1).
Animals were killed followed by brains collection out of the skull. Immediately after removal, the brain from each rat was cleaned with cold normal saline and used to make a 10% (w/v) homogenate in ice-cold 0.05 M potassium phosphate buffer (pH 7.4) using Glas-Col motor-driven homogenizer (Glas-Col Co., CA, USA), This kit uses a monoclonal antibody that binds competitively to NQO1 in the standard or samples. After incubations at room temperature immediately the excess reagents were removed and the substrate was added. When a short incubation time was finished the enzymatic reaction was blocked and the yellow color generated is read on BioTek microplate reader at 405 nm . The intensity of the yellow color is inversely proportional to the concentration of NQO1 in either standard or samples. The measured optical density is used to calculate the concentration of NQO1.
Determination of gene expression of mcu and pyk2.
RNA was extracted using (NucleoSpin®) kit. Total RNA was then extracted by using RNA Purification Kit NucleoSpin® Filter (violet ring according to the manufacturer’s instructions). NucleoSpin® RNA Column placed into a collection tube (1.5 ml, supplied with kit) then RNA eluted in 60 µl DNase –free H2O and centrifuged at 11,000 rpm for 1 min. The clarity (A260/A280 ratio) and the RNA concentration were obtained using spectrophotometry (dual-wavelength Beckman, Spectrophotometer, USA). The purified and extracted DNA samples were stored at –80 °C for further use . Target genes were detected by real-time quantitative (q) PCR (Table 1).
Table 1: Primers used in the study.
Determination Of Malonaldehyde(MDA)
Malondialdehyde (MDA) content was according to the method of Satoh 
Determination of brain myeloperoxidase (MPO) content.
The method described by Bradley et al.  was used for the determination of myeloperoxidase formation in Brain homogenate.
Determination Of Nitric Oxide (NO)
The Bio-diagnostic Nitric Oxide reagent kit was used for the determination of NO in brain homogenate according to the method described by Miranda et al. .
Determination Of Glutathione (GSH)
The level of glutathione was determined in brain homogenate using Biodiagnostic kit (Cairo, Egypt) according to the method described by Beutler et al. .
Determination of Infarction Volume.
Twenty-four hours after reperfusion animals were sacrificed and brains of whole hemisphere of the brain were collected rapidly and frozen at −80°C for 5 minutes. Brain slices were done at 2 mm from the frontal tips, and slices were dipped in 2% 2,3,5-tripenzyltetrazolium chloride (TTC) at 37 °C for 20 min. After the staining, high-quality images of these slices were taken and the software image analyzer software (Image analyzer unit, the center of mycology, Al-Azhar University, Egypt) was used to calculate the volume of infarction .
It is done according to Banchroft et al.  The brain of rats in different groups is used to make autopsy samples followed by a fixation step using 10% normal saline for 24 hr.
The data were expressed as means ± SEM. Statistical analysis was done by GraphPad Prism software (version 5, San Diego, CA, USA), The accepted probability level was less than 0.05 as statistically significant. One-way analysis of variance ANOVA was used for comparison between means followed by the Tukey-Kramer multiple comparison test.
NMD And RR Decreased the Overexpression Of MCU And Pyk2 Caused By MCAO
As shown in (Figure 1A, B) the MCAO operation in rats caused significant increases in MCU by (48.19%) and pyk2 (57.63%) after one hour of reperfusion as compared to the sham group. In contrast, administration of NMD (10 mg/kg, i.p.) for three days before MCAO significantly reduced the elevated levels of MCU and pyk2 in brain tissue by (64.15%) and (42.478%) respectively in comparison with MCAO inducted group. Similarly, treatment with RR (3 mg/kg, i.p.) for three days before MCAO significantly reduced the elevated levels of MCU and pyk2 in brain tissue by (41.16%) and (25.53%) respectively as compared to MCAO inducted group. Interestingly the combination of RR (1.5 mg/kg, i.p.) and NMD (5 mg/kg, i.p.) provides synergistic effects by reducing MCU and pyk2 content to (42.89%) and (52.75%) respectively in comparison with MCAO inducted groups.
Figure 1A,B: Effects of pretreatment with RR, NMD and their combination on brain content of gene expression of MCU and pyk2 in MCAO inducted groups. Data are expressed as mean ± SE. a,b,c are significantly different from sham group, MCAO group and combination group respectively atp < 0.05using one-way analysis of variance (ANOVA) followed by Tukey's test for multiple comparison.
NMD And RR Decreased Nqo1 And MPO Expression Induced By MCAO
The MCAO operation in rats resulted in significant increases in NQO1 by (66.6%), and MPO by (65.22%) after one hour of reperfusion as compared to the sham group. In contrast, administration of NMD (10 mg/kg, i.p.) for three days before MCAO significantly reduced the elevated levels of NQO1 and MPO in brain tissue by (55%) and (30.95%) respectively in comparison with MCAO inducted group. Similarly, treatment with RR (3 mg/kg; i.p.) for three days before MCAO significantly reduced the elevated levels of NQO1 and MPO in the brain. A combination of RR (1.5 mg/kg, i.p.) and NMD (5 mg/kg, i.p.) provides synergistic effects by reducing NQO1 and MPO content by (65.87%) and (29.9%) respectively in comparison with MCAO inducted groups (Figure 2A, B).
Figure 2A,B: Effects of pretreatment with RR, NMD and their combination on brain content of gene expression of NQO1 and MPO in MCAO inducted groups. Data are expressed as mean ± SE. a,b,c are significantly different from sham group, MCAO group and combination group respectively at p < 0.05using one-way analysis of variance (ANOVA) followed by Tukey's test for multiple comparison.
NMD And RR Reversed MDA, GSH And No Changes Induced By MCAO
The MCAO operation in rats caused significant increases in MDA by (47.12%), and NO (37.89%) but significant decrease in GSH by (37.26%) after one hour of reperfusion as compared to the sham group. In contrast, administration of NMD (10 mg/kg, i.p.) for three days before MCAO significantly reduced the elevated levels of MDA and NO in brain tissue by (28.79%) and (34.70%) respectively and significantly increased the reduced level of GSH by (37.37%) in comparison with MCAO inducted group. Similarly, treatment with RR (3 mg/kg, i.p.) for three days before MCAO significantly reduced the elevated levels of MDA and NO in brain tissue by (20.28%) and (30.54%) respectively and significantly increased the reduced level of GSH by (36.68%) in comparison with MCAO inducted group. Interestingly their combination of RR (1.5 mg/kg, i.p.) and NMD (5 mg/kg, i.p.) provides synergistic effects by reducing MDA and NO in brain tissue by (25.78%) and (35.06%) respectively and significantly increased the reduced level of GSH by (40.53%) in
Comparison with MCAO inducted group (Table 2).
Table 2: The effects of RR NMD, and their combination on brain content of MDA, GSH, NO in MCAO inducted group.
NMD And RR Decreased the Infraction Volume Induced By MCAO
The data showed that MCAO operation in rats caused significant increases in infraction volume by (96.5%) after one hour of reperfusion as compared to the sham group. In contrast, administration of NMD (10 mg/kg, i.p.) for three days before MCAO significantly reduced the elevated the infraction volume in brain tissue by (95.7%) in comparison with MCAO inducted group. Similarly, treatment with RR (3 mg/kg, i.p.) for three days before MCAO significantly reduced the elevated levels of infraction volume by (85.1%) compared to the MCAO inducted group. Interestingly their combination of RR (1.5 mg/kg, i.p.) and NMD (5 mg/kg, i.p.) provides a synergistic effect by reducing the infarction volume by (85%) in comparison with MCAO inducted groups (Figure 3). The RR, NMD, and their combination decreased the infraction volume when the brain sections are stained using a TTC stain showing the higher intensity of the red color (Figure 4).
Figure 3: Effects of pretreatment with RR, NMD and their combination on brain infraction volume in MCAOinducted groups. Data are expressed as mean ± S.E. a,b,c are significantly different from sham group, MCAO group and combination group respectively at p < 0.05using one-way analysis of variance (ANOVA) followed by Tukey's test for multiple comparison.
Figure 4: Sections of brain in the 8 groups after TTC staining of serial brain sections at 24 hours after MCAO. The percentage of infraction volume was measured by an image analyzer, which interpreted the severity of infarction.
Effect of mcao inducted group on histopathological findings of brain tissue.
Sections in the brain of animals in the sham group and dimethyl sulfonyl (DMSO) control group revealed normal tissue structure in the cerebral cortex of brain tissues (Fig.5A, B) respectively. On the other hand, the induction of MCAO revealed nuclear pyknosis, degeneration, and congestion in blood vessels in some neurons in the cerebral cortex (Fig. 5E). Treatment with nimodipine (NMD) (10mg/kg;i.p. 3days;Fig. 5C) and ruthenium red (RR) (3 mg/kg;i.p. 3days ;Fig. 5D) showed normal histopathological structure at cerebral cortex of the brain tissues compared to MCAO inducted group. Sections in the brain of animals that were injected with NMD (10mg/kg; i.p.) for three consecutive days then were inducted with MCAO revealed nuclear pyknosis and degeneration in some neurons in the cerebral cortex (Fig. 5F) In addition to treatment with ruthenium red (RR) (3mg/kg; i.p) for three consecutive days then were inducted with MCAO revealed normal histopathological structure in all parts of the brain (Fig. 5G). Treatment with a combination of RR (1.5 mg/kg; i.p.) and NMD (5mg/kg; i.p.) for three consecutive days then induction of MCAO revealed normal histopathologic structure in most of the brain parts (Figure 5H).
Figure 5: Cerebral cortex (A), (B), (C), (D), (G) and (H) are groups showed normal histological structure of the neurons. (Arrow) H&E*40. (E). MCAO group showed nuclear pyknosis and degeneration in most of the neurons with congestion in blood vessels. (Arrow) H&E*40. (F). Cerebral cortex showed nuclear pyknosis and degeneration. (Arrow) H&E*40.
Brain ischemia is one of the top causes of losing life and the third important cause of disability all over the world . In brain stroke, there is a leakage of blood delivery to brain areas which caused generation in cerebral neuronal tissues . MCAO is a classical model of cerebral ischemia . that causes regional brain infarction and neurological deficits in rats by blocking the middle cerebral artery. Middle carotid artery occlusion (MCAO) is a highly effective model in stroke induction by unilateral or bilateral occlusion . In this study, significant damage for neuronal cells, increase in inflammatory and oxidative stress markers, increase in expression of MCU/pyk2 pathway and marked histopathological alterations in the brain tissue were observed in MCAO induced rats, findings that are in agreement with previous work who confirmed that there was activation in Pyk2/MCU pathway, increase infarction volumes of the brain and reactive oxygen species in an invivo model of rat cerebral ischemi .
These results may be explained as Ca2+ overload plays an important role in ischemia-induced brain damage, MCU, located on the mitochondrial inner membrane, is the major channel responsible for mitochondrial Ca2+ uptake. Pyk2 can directly phosphorylate MCU, which enhances mitochondrial Ca2+ uptake. Thus Pyk2/MCU pathway may play a vital role in cell apoptosis and neuronal death  Also, the MCU functions as an enhancer of inflammation by increasing the calcium concentration of mitochondria and up-regulation of endoplasmic reticulum stress . In this study, it is observed that RR, NMD, and their combination specifically attenuated neuronal oxidative stress, inflammation, and decreased expressions of MCU/Pyk2 genes molecules. These results were augmented by that showing nimodipine; regulator of calcium homeostasis has a neuroprotective effect against oxidative stress and iNOS induction in the acute model of cerebral ischemia in hippocampal slices, increasing antioxidant and anti-inflammatory enzyme heme-oxygenase.
The effects of RR on neurons are still under debate. Some studies discovered the toxicity of neurons from it, while other studies found it to have a neuroprotective effect Zhao  proved that RR has neuroprotective actions during MCAO that may be attributed to its blocking action of MCU which has a crucial role in brain ischemia as illustrated previously.This study proved that the treatment with RR alone, NMD alone or their combination are effective as a protective treatment in case of cerebral brain stroke through several mechanisms. The mechanisms involved in this study are decreasing the inflammation, oxidative stress, infraction volume targeting the MCU/pyk2 pathway.
According to our results, we can conclude that RR, NMD, and their combination could ameliorate MCAO induced brain injury; which might be attributed to their potent antioxidant and anti-inflammatory activities via MCU/pyk2 pathway.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Conflicts of Interests
The authors have declared that there is no conflict of interest.
Authorship Contributions Statement
All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Furthermore, each author certifies that this material or similar material has not been submitted or published in any other journals before and all authors have approved the final article and there is no conflict of interest to be disclosed.
1. Gibson CL, Srivastava K, Sprigg N (2014) Inhibition of Rho-kinase protects cerebral barrier from ischemia-evoked injury through modulations of endothelial cell oxidative stress and tight junctions. J Neurochem 129: 816-826.
4. Donnan, G A, Fisher M, Macleod M (2008) Stroke. Lancet 371: 1612-1623.
6. Hurtado O, De Cristóbal J, Sánchez V (2003) Inhibition of glutamate release by delaying ATP fall accounts for neuroprotective effects of antioxidants in experimental stroke. The FASEB journal 17(14): 2082-2084.
8. Qiao H, Dong L, Zhang X (2012) Protective effect of luteolin in experimental ischemic stroke: upregulated SOD1, CAT, Bcl-2, and claudin-5, down-regulated MDA and Bax expression. Neurochemical research 37(9): 2014-2024.
10. Liu Y, Zhang GY, Yan JZ (2005) Suppression of Pyk2 attenuated the increased tyrosine phosphorylation of NMDA receptor subunit 2A after brain ischemia in rat hippocampus. Neurosci. Lett 379(1): 55-58.
14. Poole D P, Amadesi S, Veldhuis N A (2013) Protease-activated receptor 2 (PAR2) protein and transient receptor potential vanilloid 4 (TRPV4) protein coupling is required for sustained inflammatory signaling. Journal of Biological Chemistry 288(8): 5790-5802.
16. Behroozi-Lak T, Zarei L, Moloody–Tapeh (2017) Protective effects of intraperitoneal administration of nimodipine on ischemia-reperfusion injury in ovaries: Histological and biochemical assessments in a rat model. Journal of pediatric surgery 52(4): 602-608.
18. Zhang XL, Zheng SL, Dong FR (2012) Nimodipine improves regional cerebral blood flow and suppresses inflammatory factors in the hippocampus of rats with vascular dementia. Journal of International Medical Research 40(3): 1036-1045.
20. Amer MS, Torad FA, Shamaa A A (2016) Role of Endothelial Progenitor Cells in Management of Myocardial Infarction FollowingTotal Coronary Occlusion in Dogs. Research journal of pharmaceutical biology and chemical science 7(3): 1225-1237.
26. Banchroft JD, Stevens A, Turner DR (1996) Theory and practice of histological techniques.Fourthed. Churchil Livingstone New York, London, San Francisco, Tokyo, Japan.
27. Ahmad MA, Najmi AK, Mujeeb M (2014) Neuroprotective effect of guggulipidalone and in combination with aspirin on middle cerebral artery occlusion (MCAO) model of focal cerebral ischemia in rats. Toxicol Mech Methods 24(6): 438-47.
28. Park SJ, Nam KW, Lee HJ (2009) Neuroprotective effects of an alkaloid free ethyl acetate extract from the root of Sophoraflavescens Ait against focal cerebral ischemia in rats. Phytomedicine 16(11): 1042-51.
32. Buendia I, Tenti G, Michalska P (2017) ITH14001, a CGP37157-Nimodipine HybridDesigned to Regulate Calcium Homeostasis and Oxidative Stress, Exerts Neuroprotection in Cerebral Ischemia. ACS ChemNeurosci 18; 8(1):67-81.
33. Velasco I, Tapia R (2000) Alterations of intracellular calcium homeostasis and mitochondrial function are involved in Ruthenium Red neurotoxicity in primary cortical cultures. Journal of Neuroscience 60(4): 543-551.
34. Richter C (1998) Oxidative stress, mitochondria, and apoptosis. Restorative Neurology and Neuroscience 12: 1–4.
35. Zhao Q, Wang S, Li Y, Wang P, Li S (2013) The role of the mitochondrial calcium uniporter in cerebral ischemia/reperfusion injury in rats involves the regulation of mitochondrial energy metabolism. Molecular medicine reports 7(4): 1073-1080.