Effect of Peripheral Blood Mononuclear Cells on Induced Ischemia/Reperfusion in Skeletal Muscle of Adult Male Albino Rat: An Immunohistochemical Study

Nội dung text: Effect of Peripheral Blood Mononuclear Cells on Induced Ischemia/Reperfusion in Skeletal Muscle of Adult Male Albino Rat: An Immunohistochemical Study

1. Int.J.Curr.Microbiol.App.Sci (2020) 9(10): 2133-2144 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 10 (2020) Journal homepage: Original Research Article Effect of Peripheral Blood Mononuclear Cells on Induced Ischemia/Reperfusion in Skeletal Muscle of Adult Male Albino Rat: An Immunohistochemical Study Hend I. Abd El Halim*, Reda H. Elbakary, Ebtsam F. Okasha, Amany M. Mousa and Ehsan F. Salah Histology Department, Tanta University, Tanta, Egypt *Corresponding author ABSTRACT K e yw or ds Lower limbs ischemia is a life-threatening condition resulting from hypovolemia or vascular occlusion. Peripheral blood mononuclear cells (PBMCs) may represent a novel Ischemia treatment for ischemia reperfusion injury (IRI).This work was performed to study the /reperfusion, Skeletal muscle, effect of PBMCs on induced ischemia /reperfusion in skeletal muscle of adult male albino Peripheral blood rat. Fifty adult male albino rats were used; twenty were considered as donors and the others as recipients. The recipients were divided into three groups. Group I served as a mononuclear cells control. Group II used for induction of IRI; half of rats was sacrificed immediately Articl e Info (subgroup II- A) and the other half after 4 weeks (subgroup II-B).Group III: IRI induction was done then rats of subgroup III-A were injected with phosphate buffered saline while Accepted: rats of subgroup III-B were treated by intramuscular injection of PBMCs. Rats of group III 17 September 2020 were sacrificed after 4 weeks. Muscle samples of all groups were processed for Available Online: immunohistochemical study and morphological data were statistically analyzed. Rats 10 October 2020 treated with PBMCs showed well-evidenced repair of the skeletal muscle IRI. Introduction The rat hind limb IRI model resembles skeletal muscle ischemia in human which Peripheral vascular disease is a major health might be sometimes complicated with problem that is considered as one of the most multiple organ failure and death (Hiatt et al., frequent causes of debilitating diseases. 2015). Although the mechanisms of hind limb Lower limb ischemia is a life-threatening IRI are complicated, accumulating evidence clinical condition resulting from hypovolemia has suggested that reactive oxygen species or vascular occlusion. Reperfusion injury (ROS) and inflammation play a crucial role in occurs when re-introduction of blood is the pathogenesis of hind limb IRI (Kılıç et al., allowed to the ischemic limbs causing 2017). unfortunately ischemia/reperfusion injury (IRI) (Hynes et al., 2012; Paradis et al., Cell-based therapies have become a 2016). promising strategy for tissue regeneration. 2133
2. Int.J.Curr.Microbiol.App.Sci (2020) 9(10): 2133-2144 Peripheral blood is a large accessible source the approval of the Research Ethics of adult stem cells for both basic research and Committee in Tanta Faculty of Medicine. clinical applications. As there is a small number of mononuclear cells present in The donor rats were used for collection of peripheral blood compared to their number in peripheral blood for isolation of mononuclear bone marrow (Zhang and Huang, 2012), cells. The donor rats were injected numerous stem cells can be obtained from the subcutaneously once a day with Neupogen peripheral blood after mobilizing them from that used as a G-CSF (Amgen Inc., One bone marrow by using a granulocyte colony Amgen Center Drive, Thousand Oaks, stimulating factor (G-CSF) (Wexler et al., California 91320-1799 U.S.A. US License 2003). No. 1080) by insulin syringe at a dose of 100µg/kg for five consecutive days (Takagi et Mononuclear cells (MNCs) are a al., 2005). At the end of fifth day, the rats heterogeneous group of cells that contain were anesthetized with ether inhalation then mature T & B lymphocytes and monocytes in blood was collected by cardiac puncture after addition to a plenty of different multipotent sterilization of thorax by alcohol progenitor cells. MNCs transplantation has no (Parasuraman et al., 2010). need for in vitro culture that leads to increase the risk of contamination or repeated trials to Then, the collected blood was processed for obtain appropriate number for cell mononuclear cells isolation by density administration (Pösel et al., 2012). Until now, gradient centrifugation method using Ficoll- little information is known about the role of Hypaque. The cells were counted and injected MNCs in tissues regeneration and few studies intramuscularly for each recipient rat at a dose have been carried out to evaluate the effect of of 5×106 (Iwase et al., 2005). MNCs on muscle injuries. Subsequently, this work was done to demonstrate the effect of The recipient rats were divided into three peripheral blood mononuclear cells on groups (10 rats each) as follows skeletal muscle regeneration after induced ischemia /reperfusion injury. Group I (control group): subdivided into subgroup I-A (5 rats) from which the Materials and Methods gastrocnemius muscles were obtained without receiving any treatment and subgroup I-B (5 Animals rats)from which the gastrocnemius muscles were obtained 4 weeks after their This study was carried out on 50 adult male intramuscular injection once with 0.01mlof albino rats of average weight 200 grams; phosphate buffered saline (PBS). twenty of them considered as donors while the others were used as recipients. Rats were Group II (ischemia reperfusion injury group; housed in suitable clean properly ventilated IRI): IRI was induced by applying a rubber cages under similar conditions and fed on a tourniquet to the limb root of the right hind standard laboratory diet. They were limb in each rat for 4 hours to induce acclimatized to their environment for two ischemia, then the tourniquet was removed, weeks before starting the experiment. All the and the limb was re-perfused for 1 hour(Edna steps of the experiment were performed at the et al., 2007). Five rats were immediately Tissue Culture Unit of Histology Department, sacrificed after IRI induction and considered Faculty of Medicine, Tanta University after as a model for ischemia/reperfusion injury 2134
3. Int.J.Curr.Microbiol.App.Sci (2020) 9(10): 2133-2144 (subgroup II-A) while the other 5 rats were desired reaction was achieved. Mayer’s sacrificed after 4 weeks (subgroup II-B). hematoxylin was used as a counter stain. Group III (treated group):rats were subjected Myogenin positive cells that represent to induction of IRI as mentioned in group II. differentiating myocytes showed a brown Subgroup III-A included 5 rats and they were nuclear reaction. A section of injected IM once with 0.01mlof PBS by rhabdomyosarcoma was used as a positive insulin syringe. Subgroup III-B included 5 control and was provided by BiocareEmergo rats and they were injected locally in the right Europe. Normally, endothelial cells showed gastrocnemius muscle once with peripheral brown reaction in the nuclear membrane and blood mononuclear cells (5×106) suspended in cytoplasmic membranes to VEGF antibody. 0.01 ml PBS. In both subgroups, rats were Expression of VEGF has been shown to be up sacrificed after 4 weeks. regulated by any tissue in response to hypoxia, ischemia, hypoglycemia, Tissue processing inflammation, and malignancy. Tissue sections from kidney were used as a positive For histological study, musclespecimens were control and were provided by Abcam (Saad et fixed in 10% formalin buffered saline and al., 2005; Chen et al., 2011). Negative control processed to get paraffin sections. Serial sections were done by the same previous steps sections at 5 um thickness were cut using a without adding the primary monoclonal rabbit microtome and mounted on positive charged antibody. Finally, all the specimens were slides for immunohistochemical staining for examined and photographed using Leica light detection of myogenin and vascular microscope with built in camera at Histology endothelial growth factor (VEGF). Department, Faculty of Medicine, Tanta University. Immunohistochemical study Morphometric assessment For immunohistochemical study, sections were de-paraffinized, rehydrated and covered The image analysis was done by using the by 3% H2O2 for 10 minutes to block the software (Image J) (National Institute of activity of endogenous peroxidase to reduce Health, Bethesda, Maryland, USA). Ten nonspecific background staining. Then, they different non-overlapping randomly selected were treated with preheated citrate buffer (PH fields from each slide were quantified for: 6) for 20 minutes for antigen retrieval. Sections were incubated by (50 - 80 μL) of Number of Myogenin positive nuclei at a the primary antibody (Rabbit monoclonal magnification power of 400. antibody to Myogenin) (EP162, Biogenex, Emergo Europe) overnight in a humidified Capillary density is the number of capillaries/ chamber. Other sections were incubated by mm2 was measured at a magnification power the primary antibody (Rabbit monoclonal of 400.Capillaries were recognized as tubular antibody to VEGF) (EP1176Y, Gennova, structures positive for VEGF. Johan Gutenberg, India). After washing, the slides were covered completely by (50 - 80 Statistical analysis μL) of the secondary biotinylated goat anti- rabbit antibody. Subsequently, chromogenic Data were analyzed using one-way analysis of substrate was applied (diaminobenzidine; variance (ANOVA) followed by Turkey's test DAB) and the slides were incubated until for comparison between the groups using 2135
4. Int.J.Curr.Microbiol.App.Sci (2020) 9(10): 2133-2144 statistical package for Graph Pad Instate Statistical results software (version 3, USA). All values were expressed as means ± standard deviation. I - As regards mean number of myogenin Differences were regarded as significant if positive nuclei; there was no significant probability P-value 0.05) in the mean number of significant if P-value < 0.001 (Dawson and myogenin positive nuclei of control group Trapp, 2004). (subgroup IA, subgroup IB) and subgroup IIA. On the other hand, subgroup IIB (2.9 ± Results and Discussion 2.424) and subgroup IIIA (3 ± 2.404) depicted a significant increase (p < 0.05) compared to Immunohistochemical reaction of anti the control group. Mononuclear cells treated Myogenin rats (subgroup IIIB) (10.1 ± 3.929) revealed a highly significant increase (P value < 0.001) Both Group I (control group) (Figure 1) and in the mean number of myogenin positive subgroup II-A (Figure 2) showed negative nuclei when compared to all other subgroups. nuclear immune reactivity to myogenin while On the other hand, there was no significant subgroup II-B(Figure 3) and subgroup III- difference (p > 0.05) between subgroup IIB A(Figure 4) revealed some nuclei with and subgroup IIIA. All these data were positive immunoreactivity to myogenin. On illustrated in bar chart (1). the other hand, many nuclei with positive immunoreaction to myogenin were detected II – As regards mean number of blood in rats treated with PBMCs (subgroup III-B), capillaries, there was a highly significant the nuclei were either peripheral or central in decrease (P value < 0.001) in subgroup IIA position (Figures 5a & 5b). (60.61± 27.992) and in subgroup IIB (94.1±51.876) compared to control group. Immunohistochemical reaction of anti- There was no significance difference (p > vascular endothelial growth factor (VEGF) 0.05) in the mean number of blood capillaries in subgroup IIB compared to subgroup IIA Group I (control group) showed negative and subgroup IIIA. On the other hand, reaction in the sarcoplasm and positive mononuclear cells treated rats (subgroup IIIB) immuno reaction in the cytoplasm of (156.3 ± 56.3) revealed a highly significant endothelial cells (Figure 6).On the other hand, increase (P value < 0.001) when compared subgroup II-A showed diffuse strong positive with subgroups IIA, IIB and IIIA. The mean reaction in the form of brownish color in the number of blood capillaries in subgroup IIIB, sarcoplasm of some muscle fibers (Figure 7). that treated by MNCs, was non-significant (p Positive reaction in the cytoplasm of > 0.05) when compared to control group. All endothelial cells of some dilated blood vessels these data were illustrated in bar chart (2). (Figure 8a) in addition to localized positive reaction at the periphery of some muscle The skeletal muscle ischemia-reperfusion fibers (Figure 8b) was detected in subgroup injury (IRI) is a life-threatening metabolic II-B and subgroup III-A. Noteworthy, condition with high mortality and morbidity subgroup III-B that treated with PBMCs rates (Neto et al., 2014). Numerous showed negative reaction in the sarcoplasm intervention strategies including cell-based and appearance of the capillaries like that of therapies have been proposed aiming to control group with positive reaction in the reduce IRI (Wang et al., 2015). To the best of cytoplasm of endothelial cells (Figure 9). our knowledge, this study could be considered 2136
5. Int.J.Curr.Microbiol.App.Sci (2020) 9(10): 2133-2144 the first one searching the effect of intra- reperfusion injury of skeletal muscle in muscular injection of peripheral blood experimental animals. mononuclear cells (PBMCs) in ischemia Chart.1 Comparison between the studied groups as regard means ± SD of myogenin positive nuclei Chart.2 Comparison between the studied groups as regard means ± SD of capillary density Fig.1 A longitudinal section in the gastrocnemius muscle of a control rat showing negative nuclear reaction (arrows) (Myogenin immunoreaction counterstained with Hx, x1000) 2137
6. Int.J.Curr.Microbiol.App.Sci (2020) 9(10): 2133-2144 Fig.2 A longitudinal section in the gastrocnemius muscle of a rat in subgroup (II-A) showing negative nuclear reaction (arrows) (Myogenin immunoreaction counterstained with Hx, x1000) Fig.3 A longitudinal section in the gastrocnemius muscle of a rat in subgroup (II-B) showing some dark brown myogenin positive nuclei (arrows) (Myogenin immunoreaction counterstained with Hx, x1000) Fig.4 A longitudinal section in the gastrocnemius muscle of a rat in subgroup (III-A) showing some myogenin positive nuclei (arrows) (Myogenin immunoreaction counterstained with Hx, x1000) 2138
7. Int.J.Curr.Microbiol.App.Sci (2020) 9(10): 2133-2144 Fig.5 Longitudinal sections in the gastrocnemius muscles of subgroup (III-B) showing numerous positive nuclei (arrows) arranged in rows either peripherally (A) or centrally (B) located (Myogenin immunoreaction counterstained with Hx, x1000) Fig.6 A transvers section in the gastrocnemius muscle of a control rat showing negative immunoreactivity in myofibers (stars) and positive reaction in the cytoplasm of endothelial cells (arrows) (VEGF immunoreaction counterstained with Hx, x1000) Fig.7 A section in the gastrocnemius muscle obtained from a rat in subgroup (II-A) showing diffuse strong positive brown immunoreactivity in the sarcoplasm of muscle fibers (arrows) (VEGF immunoreaction counterstained with Hx, x1000) 2139
8. Int.J.Curr.Microbiol.App.Sci (2020) 9(10): 2133-2144 Fig.8 Sections in the gastrocnemius muscle of subgroup (II-B) showing (A): localized positive immunoreactivity at the periphery of some muscle fibers (arrows). (B): a positive reaction in the cytoplasm of endothelial cells (arrows) (VEGF immunoreaction counterstained with Hx, x1000) Fig.9 A section in the gastrocnemius muscle of a rat in subgroup (III-B) showing a negative reaction in the sarcoplasm (stars) with positive reaction in the cytoplasm of endothelial cells (arrows) (VEGF immunoreaction counterstained with Hx, x1000) In this study, differentiation of satellite cells myogenic cell pool necessary for myofibers was detected using myogen in formation. Also, Youssef et al., (2015) and immunostaining that revealed a significant Nada et al., (2018) observed positive nuclei increase in the mean number of myogen in for myogenin and explained it as immuno reacting cells in PBMCs treated rats differentiating satellite cells overlapping the (subgroup III-B) when compared with control myofibers. group and IRI group. This was in accordance with the work of Christov et al., (2007) who Moreover, Faralli and Dilworth (2012) stated reported that PBMCs produce more that muscular injury activates satellite cells to pronounced increase of myogenic precursor divide and differentiate to specialized cells together with an expansion of the myogenic precursor cells expressing 2140
9. Int.J.Curr.Microbiol.App.Sci (2020) 9(10): 2133-2144 myogenin in attempt to repair the damaged higher number of capillaries in comparison muscle. In addition, Yin et al., (2013) and with IRI group that was expressed by the term Wilschut et al., (2015) reported that capillary density. The capillary density was myogenin is a specific gene that is essential counted as number of capillaries per mm2 for muscle development and regeneration. according to a previous study done by Usui et They added that, myogenin expression is al., (2014). Similarly, Wang et al., (2015) restricted to cells of skeletal muscle origin but found that the capillary density in ischemic it is not detected in adult skeletal muscles. In limbs was significantly greater in the PBMCs addition, myogenin is an important indicator treated group than in the non-treated groups of differentiation of satellite cells. as evident using VEGF immunoreaction in the present study. In the present study, immunohistochemical staining for detection of VEGF in IRI group The therapeutic effect of PBMCs could be revealed a diffuse strong positive reaction in explained by Rehman et al., (2003) who the sarcoplasm of ischemic muscle fibers in suggested that PBMCs promote angiogenesis immediate sacrificed rats after induction by activation of endothelial progenitor cell (subgroup II-A). Meanwhile, the rats that migration to the ischemic region and secrete were left for four weeks without treatment several angiogenic factors that promote (subgroup II-B) showed a localized positive neovascularization in addition to cytokines, reaction at the periphery of myofibers fibroblast growth factors and other factors sarcoplasm in addition to positive reaction in that inhibit apoptosis. the cytoplasm of endothelial cells of dilated blood vessels. These findings were in In addition, Costa-Rodrigues et al., (2010) accordance with the work of Rissanen et al., and Zhang and Huang (2012) reported that, (2002) who noticed that diffuse expression of mononuclear cells have the potential to VEGF occurs in acute ischemia while in differentiate into cell types other than chronic skeletal muscle ischemia VEGF phagocytes including; endothelial cells, expression is restricted to atrophic and hepatocytes, cardiomyogenic cells, smooth regenerating muscle cells. muscle cells, osteoblasts, osteoclasts, epithelial cells, neural cells, fat cells and In addition, Louboutin et al., (2012) stated skeletal muscle cells. that VEGF is found on the cell membrane, cytoplasm and nuclei of regenerating and Moreover, Lee (2014) and Beer et al., (2016) atrophic skeletal muscle fibers. Moreover, added that PBMCs preparations, without any Zhang et al., (2014) reported that VEGF is an in-vitro manipulation, could be useful for angiogenic growth factor that was expressed tissue regeneration, because they contain in response to tissue ischemia and its role is immunomodulatory lymphocytes expressing mainly restricted to angiogenesis and various cytokines and paracrine factors called endothelial cell growth. the cell secretomes that exert beneficial biological effects and promote tissue Interestingly, in the present study peripheral regeneration. On the other hand, Ceafalan et blood mononuclear cells treated rats al., (2018) stated that the implanted (subgroup III-B) showed many blood mononuclear cells do not secrete angiogenic capillaries in the form of tubular structures factors sufficient for neovascularization but with brownish endothelial cells in VEGF instead stimulate muscle cells to produce immunoreaction. PBMCs treated rats revealed angiogenic factors, thereby promoting 2141
10. Int.J.Curr.Microbiol.App.Sci (2020) 9(10): 2133-2144 neovascularization in ischemic tissues. Chazaud, B.,and Gherardi, RK. 2007. From the previous data, it was concluded that Muscle satellite cells and endothelial PBMCs had a significant effect on cells: close neighbors and privileged enhancement of muscle regeneration after partners. Molecular Biology of the Cell; IRI. This effect was obtained by promoting (4): 1397-1409. proliferation, activation and differentiation of Costa-Rodrigues, J., Teixeira, CA., Sampaio, satellite cells in addition to neovascularization P., and Fernandes, MH. 2010. and formation of multiple capillaries. Characterisation of the osteoclastogenic Accordingly, using PBMCs as a promising potential of human osteoblastic and treatment option to promote skeletal muscle fibroblastic conditioned media. J Cell repair in peripheral arterial disease can Biochem; 109: 205-216. become a reality in the near future. Dawson, B., and Trapp, R.2004. Research questions about relationships among Acknowledgments variables. (Ch.8). In basic & clinical biostatistics. The McGraw-Hill There is no conflict of interest. This research Companies. USA; pp 190-220. did not receive any specific grant from Edna, M., Maeli, D., Vitalino, P., Rubens, C., funding agencies in the public, commercial, and Ana Lucia, A. 2007. Ischemia and or not-for-profit sectors. reperfusion effects on skeletal muscle tissue: morphological and histochemical Declaration of interest studies. Int J Exp Path; 88: 147–154. Faralli, H., and Dilworth, FJ.2012. Turning on All authors declare no related conflicts of Myogenin in Muscle: A Paradigm for interest. Understanding Mechanisms of Tissue- Specific Gene Expression. Comp Funct References Genomics; 2012: 836374. Hiatt, WR., Armstrong, EJ., Larson, CJ., and Beer, L., Mildner, M., Gyöngyösi, M.,and Brass, EP. 2015. Pathogenesis of the Ankersmit, HJ. 2016. Peripheral blood limb manifestations and exercise mononuclear cell secretome for tissue limitations in peripheral artery disease. repair. Apoptosis; 21(12): 1336-1353. Circulation Research; 116(9): 1527– Ceafalan, LC., Fertig, TE., Popescu, AC., 1539. Popescu, BO., Hinescu, ME., and Hynes, BG., Margey, RJ., Ruggiero Ii, N., Gherghiceanu, M. 2018. Skeletal muscle Kiernan, TJ., Rosenfield, K, and Jaff, regeneration involves macrophage- MR. 2012. Endovascular management myoblast bonding. Cell Adhesion & of acute limb ischemia. Annals of Migration; 12(3): 228-235. Vascular Surgery; 26(1): 110-124. Chen, L., Huang, HW., Gu, SH., Xu, L., Gu, Iwase, T., Nagaya, N., Fujii, T., Itoh, T., YD., Xu, JG., and Xu, JG.2011. The Murakami, S., Matsumoto, T., study of myogenin expression in Kangawa, K., and Kitamura, S.2005. denervated human skeletal muscles. Comparison of angiogenic potency Journal of International Medical between mesenchymal stem cells and Research; 39(2): 378-387. mononuclear cells in a rat model of Christov, C., Chrétien, F., Abou-Khalil, R., hindlimb ischemia. Cardiovascular Bassez, G., Vallet, G., Authier, FJ., Research; 66(3): 543-551. Bassaglia, Y., Shinin, V., Tajbakhsh, S., Kılıç, Y., Özer, A., Tatar, T., Zor, 2142