Ratlarda Deneysel Spinal Kord Hasar Modelinde Genisteinin Nöroprotektif Etkisinin Araştırılması, Diffüz Tensor Görüntüleme ile Değerlendirilmesi

Yıl 2020, Sayı: 11, 130 - 149, 31.08.2020

Gülşah Öztürk , Gökalp Silav Said İncir Ayça Arslanhan Mustafa Ali Akçetin Orkun Zafer Toktaş Deniz Konya

https://doi.org/10.38079/igusabder.742525

Öz

Amaç: Spinal kord hasarı (SKH), insidansı çok yüksek olmamasına rağmen, bu durumun sonuçları birey, aile ve toplum için son derece önemli sekellerle sonuçlanabilecek bir hastalıktır. Nöral hasar onarımı ile ilgili her geçen gün yeni çalışmalar umut vadetmekle birlikte SKH için kür olabilecek altın standart bir tedavi henüz yoktur. Bu çalışmada ratlarda ağırlık düşürme modeli kullanılarak oluşturulan spinal kord hasarı sonrası tedavi amaçlı verilen; bir tirozinkinaz inhibitörü olan Genistein (GEN) isimli fitoöstrojenin etkisi araştırılmış ve geç dönemde sayılı rat üzerindeki diffüzyon tensör görüntüleme (DTG) ile sonuçları değerlendirilmiştir. 

Yöntem: Çalışma Marmara Üniversitesi Başıbüyük Nörolojik Bilimler Enstitüsü’nde yapıldı. Çalışmada toplam 28 adet 200-250 gr ağırlığında Sprague-Dawley sıçan randomize olarak 4 eşit gruba bölündü: Grup 1 (n=7 sıçan) kontrol grubu (sadece laminektomi uygulanan), Grup 2 (n=7 sıçan) travma grubu, Grup 3 (n=7 sıçan) travma + dimetil sülfoksid (DMSO) uygulanmış grup, Grup 4 (n=7 sıçan) travma+DMSO+GEN (0,25 mg /kg/rat GEN) uygulanmış grup. Genistein DMSO aracılığıyla çözülebilen bir maddedir. Grup 1’e sadece T10-12 laminektomi uygulandı. Grup 2, 3 ve 4’e T10-12 total laminektomi sonrası ağırlık düşürme modeli kullanılarak spinal kord travması yapıldı. Grup 4’e 7 gün boyunca 0,25 mg/kg/rat GEN uygulandı. Grup 1’den bir, diğer gruplardan 3’er adet toplam 10 adet randomize seçilen ratın spinal kordu postoperatif 28. günde Diffüzyon tensör görüntüleme ile değerlendirildi. Alınan doku örnekleri Hematoksilen-Eosin (HE), Kristal Viole ve Luksol Fast Blue (LFB) ile boyanıp ışık mikroskobunda incelendi. Çalışmada kullanılan tüm ratlar postoperatif 6. saat, 24. saat, 7., 14., 21. ve 28. günlerde lökomotor derelecelendirme skalası (BBB) kullanılarak değerlendirildi.

Bulgular: GEN grubunda, diğer travma gruplarına göre fonksiyonel iyileşme puanları daha iyi olmasına rağmen tüm travma grupları arasında anlamlı istatistiksel fark gözlenmemiştir (p>0.05). Travmadan 28 gün sonra alınan görüntü örneklerinde, travma uygulanan gruplarda, lezyon merkezinde fraksiyonel anizotropi (FA) değerlerinin azaldığı gözlenmiştir.

Sonuç: Spinal kord travmasında GEN etkinliği, GEN uygulanmış travma grubunda, diğer travma gruplarıyla karşılaştırıldığında nörolojik iyileşmede BBB motor skala sonuçlarına göre artış göstermiş olup istatistiksel olarak anlamlı sonuç elde edilmemiştir. Çalışma, spinal kord travmasında GEN kullanımı için temel bir bilgi düzeyi oluşturmuş olup daha geniş kapsamlı bir çalışmada doz bağımlı araştırma yapılabilir. Yardımcı tanısal araç olarak kullanılan DTG’nin travma sonrası takipte önemli rol alabileceği öngörülmüştür. 

Anahtar Kelimeler

diffuz tensor görüntüleme, spinal kord hasarı, genistein, fraksiyonel anizotropi, rat, traktografi, omurilik travması

Investigation of Neuroprotective Effect of Genistein in Experimental Spinal Cord Injury Model in Rats, Evaluation with Diffuse Tensor Imaging

Öz

Aim: Spinal cord injury (SCI) is a wide spectrum of a devastating disease which has significant effects on individuals, families or society even though the incidence is low. Studies on neural tissue repair have huge progress in recent years but spinal cord injury still remains a troublesome condition with no definitive cure. In this study, we aimed to explore the effectiveness of Genistein (GEN), a phytoestrogen and tyrosine kinase inhibitor in a rat thoracic weight drop SCI model and to evaluate the long term results with diffusion tensor imaging (DTI), locomotion and histopathological changes.

Method: This study was conducted at Marmara University Basıbuyuk Neurological Sciences Institute. In our study twenty-eight, 250 g weighted, young adult Sprague-Dawley rats were used. They were randomly divided into four equivalent groups: 1.group Sham (n=7) 2. group Experimental spinal cord injury (SCI) (n=7) 3. group SCI + dimethyl sulfoxide (DMSO) (n=7) 4. group SCI+ DMSO+ GEN (0,25 mg /kg/rat GEN)(n=7). In the first sham group, only T10-12 laminectomy was done. Spinal cord trauma was performed in Group 2, 3 and 4 using the weight-drop model after T10-12 total laminectomy. GEN was dissolved in the vehicle DMSO. SCI+ DMSO+ GEN rat model: GEN (0,25 mg /kg/rat GEN) were given to rats subjected to SCI for 7 days. One rat from the first group, three from the 2nd group, three from 4th group, in total 10 rats were randomly chosen and magnetic resonance imaging (MRI) performed with DTI at 28th day. Tissue samples were stained with Hematoxylin-Eosin (HE), Crystal Viole and Luxol Fast Blue (LFB) and examined under a light microscope. The Basso-Beattie-Bresnehan (BBB) locomotor rating scale was performed at 6th hour, 24th hour and weekly for four weeks for all the rats.

Results: In GEN group, there was a higher functional improving but, there was no statistical significiantly differences (p>0.05) between all trauma groups. At 28th day of the injury, we took images of all groups which had been chosen randomly and we found that Fractional Anisotropy (FA) decreased at the epicenter zone.

Conclusion: According to BBB motor scale results in terms of effectiveness of GEN in the spinal cord injury; although compared to the other groups, we obtained increased neurological recovery. Results were not statistically significant. Study creates a basic level of knowledge about the GEN dosage and activity in the spinal cord injury, but a more comprehensive dose-dependent research study should be performed. DTI seems as a helpful diagnostic tool in study and it can take an important role on follow-up after trauma.

Anahtar Kelimeler

spinal cord injury, genistein, diffusion tensor imaging, fractional anisotropy, tractography, rat, dti

Kaynakça

• DeVivo MJ, Krause JS, Lammertse DP. Recent trends in mortality and causes of death among persons with spinal cord injury. Arch Phys Med Rehabil. 1999;80(11):1411-1419.
• Lee BB, Cripps RA, Fitzharris M, Wing PC. The global map for traumatic spinal cord injury epidemiology: update 2011, global incidence rate. Spinal Cord. 2014;52(2):110-116.
• McKinley WO, Jackson AB, Cardenas DD, DeVivo MJ. Long-term medical complications after traumatic spinal cord injury: a regional model systems analysis. Arch Phys Med Rehabil. 1999;80(11):1402-1410.
• Ramer MS, Harper GP, Bradbury EJ. Progress in spinal cord research - a refined strategy for the International Spinal Research Trust. Spinal Cord. 2000;38(8):449-472.
• Del Bigio MR, Johnson GE. Clinical presentation of spinal cord concussion. Spine (Phila Pa 1976). 1989;14(1):37-40.
• Wilberger JE. The Merck Manuels: The Merck Manuel for Healthcare Professionals; 2011.
• Hilton BJ, Moulson AJ, Tetzlaff W. Neuroprotection and secondary damage following spinal cord injury: concepts and methods. Neurosci Lett. 2017;652:3-10.
• Zwimpfer TJ, Bernstein M. Spinal cord concussion. J Neurosurg. 1990;72(6):894-900.
• Griffin JM, Bradke F. Therapeutic repair for spinal cord injury: combinatory approaches to address a multifaceted problem. EMBO Mol Med. 2020;12(3):e11505.
• Joaquim AF, Daniel JW, Schroeder GD, Vaccaro AR. Neuroprotective agents as an adjuvant treatment in patients with acute spinal cord injuries: a qualitative systematic review of randomized trials. Clin Spine Surg. 2020;33(2):65-75.
• Bracken MB. Steroids for acute spinal cord injury. Cochrane Database Syst Rev. 2012;1:CD001046.
• Young W, Bracken MB. The second national acute spinal cord injury study. J Neurotrauma. 1992;9(Suppl 1):S397-405.
• Bracken MB, Shepard MJ, Collins WF, et al. A randomized, controlled trial of methylprednisolone or naloxone in the treatment of acute spinal-cord injury. Results of the second national acute spinal cord injury study. N Engl J Med. 1990;322(20):1405-1411.
• Bracken MB, Aldrich EF, Herr DL, et al. Clinical measurement, statistical analysis, and risk-benefit: controversies from trials of spinal injury. J Trauma. 2000;48(3):558-561.
• Evaniew N, Belley-Cote EP, Fallah N, Noonan VK, Rivers CS, Dvorak MF. Methylprednisolone for the treatment of patients with acute spinal cord injuries: a systematic review and meta-analysis. J Neurotrauma. 2016;33(5):468-481.
• Liu LX, Chen WF, Xie JX, Wong MS. Neuroprotective effects of genistein on dopaminergic neurons in the mice model of Parkinson's disease. Neurosci Res. 2008;60(2):156-161.
• McClain RM, Wolz E, Davidovich A, Pfannkuch F, Bausch J. Subchronic and chronic safety studies with genistein in dogs. Food Chem Toxicol. 2005;43(10):1461-1482.
• Tsai TH. Concurrent measurement of unbound genistein in the blood, brain and bile of anesthetized rats using microdialysis and its pharmacokinetic application. J Chromatogr A. 2005;1073(1-2):317-322.
• McDowell ML, Das A, Smith JA, Varma AK, Ray SK, Banik NL. Neuroprotective effects of genistein in VSC4.1 motoneurons exposed to activated microglial cytokines. Neurochem Int. 2011;59(2):175-184.
• Ismail M, Ibrahim S, El-Amir A, El-Rafei AM, Allam NK, Abdellatif A. Genistein loaded nanofibers protect spinal cord tissue following experimental injury in rats. Biomedicines. 2018;6(4):96.
• Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma. 1995;12(1):1-21.
• Bracken MB. Methylprednisolone and spinal cord injury. J Neurosurg. 2000;93(1 Suppl):175-179.
• Sribnick EA, Samantaray S, Das A, et al. Postinjury estrogen treatment of chronic spinal cord injury improves locomotor function in rats. J Neurosci Res. 2010;88(8):1738-1750.
• Kachadroka S, Hall AM, Niedzielko TL, Chongthammakun S, Floyd CL. Effect of endogenous androgens on 17beta-estradiol-mediated protection after spinal cord injury in male rats. J Neurotrauma. 2010;27(3):611-626.
• Ritz MF, Hausmann ON. Effect of 17beta-estradiol on functional outcome, release of cytokines, astrocyte reactivity and inflammatory spreading after spinal cord injury in male rats. Brain Res. 2008;1203:177-188.
• Morrow AL, Biggio G, Serra M, et al. The role of neuroactive steroids in ethanol/stress interactions: proceedings of symposium VII at the Volterra conference on alcohol and stress. Alcohol. 2009;43(7):521-530.
• Simpkins JW, Singh M, Brock C, Etgen AM. Neuroprotection and estrogen receptors. Neuroendocrinology. 2012;96(2):119-130.
• Kuiper GG, Lemmen JG, Carlsson B, et al. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology.1998;139(10):4252-4263.
• Han S, Wu H, Li W, Gao P. Protective effects of genistein in homocysteine-induced endothelial cell inflammatory injury. Mol Cell Biochem. 2015;403(1-2):43-49.
• Fotsis T, Pepper MS, Montesano R, et al. Phytoestrogens and inhibition of angiogenesis. Baillieres Clin Endocrinol Metab. 1998;12(4):649-666.
• Kousidou O, Tzanakakis GN, Karamanos NK. Effects of the natural isoflavonoid genistein on growth, signaling pathways and gene expression of matrix macromolecules by breast cancer cells. Mini Rev Med Chem. 2006;6(3):331-337.
• Fuhrman B, Aviram M. Flavonoids protect LDL from oxidation and attenuate atherosclerosis. Curr Opin Lipidol. 2001;12(1):41-48.
• McConkey DJ, Orrenius S. The role of calcium in the regulation of apoptosis. Biochem Biophys Res Commun. 1997;239(2):357-366.
• Sribnick EA, Ray SK, Nowak MW, Li L, Banik NL. 17beta-estradiol attenuates glutamate-induced apoptosis and preserves electrophysiologic function in primary cortical neurons. J Neurosci Res. 2004;76(5):688-696.
• Das A, McDowell M, Pava MJ, et al. The inhibition of apoptosis by melatonin in VSC4.1 motoneurons exposed to oxidative stress, glutamate excitotoxicity, or TNF-alpha toxicity involves membrane melatonin receptors. J Pineal Res. 2010;48(2):157-169.
• Fotsis T, Pepper M, Adlercreutz H, et al. Genistein, a dietary-derived inhibitor of in vitro angiogenesis. Proc Natl Acad Sci U S A. 1993;90(7):2690-2694.
• An J, Tzagarakis-Foster C, Scharschmidt TC, Lomri N, Leitman DC. Estrogen receptor beta-selective transcriptional activity and recruitment of coregulators by phytoestrogens. J Biol Chem. 2001;276(21):17808-17814.
• Duffy C, Perez K, Partridge A. Implications of phytoestrogen intake for breast cancer. CA Cancer J Clin. 2007;57(5):260-277.
• Liu J, Xu K, Wen G, et al. Comparison of the effects of genistein and zoledronic acid on the bone loss in OPG-deficient mice. Bone. 2008;42(5):950-959.
• Groyer G, Eychenne B, Girard C, Rajkowski K, Schumacher M, Cadepond F. Expression and functional state of the corticosteroid receptors and 11 beta-hydroxysteroid dehydrogenase type 2 in Schwann cells. Endocrinology. 2006;147(9):4339-4350.
• Incir S, Bolayirli IM, Inan O, et al. The effects of genistein supplementation on fructose induced insulin resistance, oxidative stress and inflammation. Life Sci. 2016;158:57-62.
• Ford JC, Hackney DB, Alsop DC, et al. MRI characterization of diffusion coefficients in a rat spinal cord injury model. Magn Reson Med. 1994;31(5):488-494.
• Ellingson BM, Salamon N, Holly LT. Imaging techniques in spinal cord injury. World Neurosurg. 2014;82(6):1351-1358.
• Schwartz ED, Hackney DB. Diffusion-weighted MRI and the evaluation of spinal cord axonal integrity following injury and treatment. Exp Neurol. 2003;184(2):570-589.
• Ellingson BM, Ulmer JL, Kurpad SN, Schmit BD. Diffusion tensor MR imaging in chronic spinal cord injury. AJNR Am J Neuroradiol. 2008;29(10):1976-1982.
• Ellingson BM, Ulmer JL, Kurpad SN, Schmit BD. Diffusion tensor MR imaging of the neurologically intact human spinal cord. AJNR Am J Neuroradiol. 2008;29(7):1279-1284.
• Demir A, Ries M, Moonen CT, et al. Diffusion-weighted MR imaging with apparent diffusion coefficient and apparent diffusion tensor maps in cervical spondylotic myelopathy. Radiology. 2003;229(1):37-43.
• Ellingson BM, Kurpad SN, Schmit BD. Ex vivo diffusion tensor imaging and quantitative tractography of the rat spinal cord during long-term recovery from moderate spinal contusion. J Magn Reson Imaging. 2008;28(5):1068-1079.
• Ellingson BM, Kurpad SN, Li SJ, Schmit BD. In vivo diffusion tensor imaging of the rat spinal cord at 9.4T. J Magn Reson Imaging. 2008;27(3):634-642.
• Ellingson BM, Ulmer JL, Schmit BD. Morphology and morphometry of human chronic spinal cord injury using diffusion tensor imaging and fuzzy logic. Ann Biomed Eng. 2008; 36(2):224-236.
• Ellingson BM, Ulmer JL, Prost RW, Schmit BD. Morphology and morphometry in chronic spinal cord injury assessed using diffusion tensor imaging and fuzzy logic. Conf Proc IEEE Eng Med Biol Soc. 2006;2006:1885-1888.
• Ellingson BM, Schmit BD, Kurpad SN. Lesion growth and degeneration patterns measured using diffusion tensor 9.4-T magnetic resonance imaging in rat spinal cord injury. J Neurosurg Spine. 2010;13(2):181-192.
• Deo AA, Grill RJ, Hasan KM, Narayana PA. In vivo serial diffusion tensor imaging of experimental spinal cord injury. J Neurosci Res. 2006;83(5):801-810.
• Madi S, Hasan KM, Narayana PA. Diffusion tensor imaging of in vivo and excised rat spinal cord at 7 T with an icosahedral encoding scheme. Magn Reson Med. 2005;53(1):118-125.
• Lee JW, Kim JH, Park JB, et al. Diffusion tensor imaging and fiber tractography in cervical compressive myelopathy: preliminary results. Skeletal Radiol. 2011;40(12):1543-1551.
• Motovylyak A, Skinner NP, Schmit BD, Wilkins N, Kurpad SN, Budde MD. Longitudinal in vivo diffusion magnetic resonance imaging remote from the lesion site in rat spinal cord injury. J Neurotrauma. 2019;36(9):1389-1398.
• Kelley BJ, Harel NY, Kim CY, et al. Diffusion tensor imaging as a predictor of locomotor function after experimental spinal cord injury and recovery. J Neurotrauma. 2014;31(15):1362-1373.
• Pease A, Miller R. The use of diffusion tensor imaging to evaluate the spinal cord in normal and abnormal dogs. Vet Radiol Ultrasound. 2011;52(5):492-497.
• Wang F, Huang SL, He XJ, Li XH. Determination of the ideal rat model for spinal cord injury by diffusion tensor imaging. Neuroreport. 2014;25(17):1386-1392.
• Wang-Leandro A, Hobert MK, Kramer S, Rohn K, Stein VM, Tipold A. The role of diffusion tensor imaging as an objective tool for the assessment of motor function recovery after paraplegia in a naturally-occurring large animal model of spinal cord injury. J Transl Med. 2018;16(1):258.