Tavşanda Spinal Kord İskemi/Reperfüzyon Hasarı Modelinde Tiagabinin Nöroprotektif Etkileri

Öz

Amaç: Omurilik iskemi/reperfüzyon (İ/R) hasarı ciddi bir klinik sorundur. Reperfüzyon fazı, aşırı glutamat salınımına yol açarak nöronal hasara neden olan toksik koşullar oluşturur. Tiagabin, gama-aminobütirik asit (GABA) geri alım sürecini spesifik olarak bloke ettiği için GABA taşıyıcı-1 (GAT-1) inhibitörü olarak işlev görür. GABAerjik iletimi arttırarak çeşitli merkezi sinir sistemi hasarı modellerinde nöroprotektif etkiler gösterdiği bilinmektedir. Önceki çalışmalar Tiagabin’in nöroprotektif etkilerini göstermiştir ancak daha hiçbir çalışma Tiagabin’in omurilik iskemi/reperfüzyon (İ/R) hasarı üzerindeki etkisini incelememiştir. Bu çalışmada, deneysel bir tavşan omurilik İ/R hasarı modelinde Tiagabin’in nasıl bir nöroprotektif etkiye sahip olduğunu araştırmayı amaçladık. Gereç ve Yöntemler: Toplam 50 tavşan, 10 tavşandan oluşan 5 gruba rastgele dağıtıldı: Grup 1 (control), Grup 2 (İ/R) ve gösterildiği gibi tedavi grupları; Grup 3 (10 mg/kg doz Tiagabin ile İ/R), Grup 4 (20 mg/kg doz Tiagabin ile İ/R) ve Grup 5 (40 mg/kg doz Tiagabin ile İ/R). Kontrol grubunda sadece laparatomi uygulandı. Diğer tüm gruplarda, omurilik iskemi modeli, renal arterin hemen kaudalindeki aortun travmatik olmayan bir vasküler klemp ile 45 dakika süreyle oklüzyonuyla oluşturuldu. Oklüzyon süresinden sonra klemp çıkarıldı ve reperfüzyon başlatıldı. Tavşanlar 48 saatlik reperfüzyon süresi boyunca bireysel kafeslerde takip edildi. Tiagabin uygulaması, iskemi başlatılmadan 30 dakika önce intraperitoneal enjeksiyon yoluyla gerçekleştirildi. Apoptotik hücreleri belirlemek için TUNEL boyaması ile birlikte histopatolojik değerlendirme ve oksidatif stres göstergelerinin biyokimyasal değerlendirmelerini gerçekleştirdik. Malondialdehit (MDA), katalaz (CAT), süperoksit dismutaz (SOD) ve glutatyon peroksidaz (GSH-Px) düzeyleri ile oksidatif stres parametreleri analiz edildi. Çalışma TNF-α and IL-1β ve IL-6’yı içeren inflamatuar sitokinlerin ve kaspaz-3 aktivitesinin ölçümlerini de içeriyordu. Nörolojik değerlendirme, Tarlov skorlama sistemi kullanılarak 48 saatlik reperfüzyonda yapıldı. Bulgular: Tiagabin alan gruplar, İ/R grubuna göre daha iyi nörolojik iyileşme gösterdi. İ/R grubunda parapleji oranı %80 olarak belirlenirken, 40 mg/kg Tiagabin uygulanan grupta bu oran %10’a düştü (p<0.05). Tiagabin tedavisi, GABA seviyelerini arttırırken glutamate konsantrasyonlarını azalttı. Çalışmamız, MDA ve proinflamatuar sitokinlerin doz artışıyla orantılı olarak azaldığını, ancak antioksidan enzim aktivitelerinin arttığını gösterdi. Bax-Bcl-2 oranı ve kaspaz-3 ekspresyonu, apoptotic belirteçlerde önemli bir azalma gösterdi. Tedavi grubundaki sağlam motor nöron sayısı, kontrol grubundakinin %77’sine ulaştı. TUNEL pozitif hücre sayımları açısından, 4. grupta alan başına 24.36±4.28 hücre varken, 2. Grupta 48.72±7.86 hücre vardı (p<0.05). Sonuç: Sonuçlarımız, Tiagabin’nin omurilik İ/R hasarına karşı güçlü bir nöroprotektif etki gösterdiğini ortaya koymuştur. Koruma sistemi, oksidatif stresi azaltmak, inflamasyonu kontrol etmek ve hücre ölümünü önlemek için üç ana mekanizma içeren tek bir savunma sistemi yerine birden fazla kanal aracılığıyla çalışmaktadır. Reperfüzyon fazı, glutamat kaynaklı eksitotoksisiteyi dengelediği için Tiagabin’nin neden olduğu GABAerjik iletimden fayda görmektedir. Bulgularımız, GAT-1’i bloke etmeni bu tür hasarlanmaların tedavisinde terapötik bir yaklaşım olarak kullanılabileceğini göstermektedir.

Anahtar Kelimeler

• Nöroproteksiyon
• Tiagabin
• Omurilik
• İskemi/Reperfüzyon hasarı

NEUROPROTECTIVE EFFECTS OF TIAGABINE IN A RABBIT MODEL OF SPINAL CORD ISCHEMIA/ REPERFUSION INJURY

Öz

Objective: Spinal cord ischemia/reperfusion (I/R) injury is a serious clinical problem. The reperfusion phase leads to excessive glutamate release which produces toxic conditions that result in neuronal damage. Tiagabine functions as a gamma-aminobutyric acid (GABA) transporter 1 (GAT-1) inhibitor because it specifically blocks the process of GABA reuptake. It is known to exhibit neuroprotective effects in various central nervous system injury models by enhancing GABAergic transmission. Previous studies demonstrated the neuroprotective effects of Tiagabine, but no previous study has examined of Tiagabine on spinal cord ischemia/reperfusion (I/R) injury. In this study, we aimed to investigate whether how Tiagabine has a neuroprotective effect in an experimental rabbit spinal cord I/R injury model. Material and Methods: A total of 50 rabbits were randomised into five groups of ten animals as follows: group 1 (control), group 2 (I/R), and treatment groups as shown; group 3 (I/R with 10 mg/kg dose Tiagabine), group 4 (I/R with 20 mg/kg dose Tiagabine) and group 5 (I/R with 40 mg/kg dose Tiagabine). In the control group only a laparotomy was performed. In all other groups, the spinal cord ischemia model was created by the occlusion of the aorta just caudal to the renal artery with an atraumatic vascular clamp for 45 minutes. After the occlusion period, the clamp was removed and reperfusion was initiated. Rabbits were followed in individual cages for 48 hours of reperfusion. We performed Tiagabine administration through intraperitoneal injection 30 minutes before the start of ischemia. We conducted histopathological evaluation along with TUNEL staining to identify apoptotic cells and biochemical assessments of oxidative stress indicators. Levels of malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were analysed, as were the oxidative stress parameters. The study included measurements of inflammatory cytokines which included TNF-α and IL-1β and IL-6 and caspase-3 activity. Neurological evaluation was assessed at 48 hours reperfusion using the Tarlov scoring system. Results: The groups which received Tiagabine showed better neurological recovery than the I/R group. The paraplegia rate was determined to be 80% in the I/R group, while this rate decreased to 10% in the group administered 40 mg/kg Tiagabine (p<0.05). The treatment of Tiagabine led to increased GABA levels while it reduced glutamate concentrations. Our study showed that MDA and proinflammatory cytokines decreased in proportion to the dose increase but antioxidant enzyme activities rose. The Bax/Bcl-2 ratio and caspase-3 expression showed substantial reduction in apoptotic markers. The number of intact motor neurons in the treatment group reached 77% of that in the control group. Regarding TUNEL-positive cell counts, group 4 had 24.36±4.28 cells per field, while the group 2 had 48.72±7.86 cells (p<0.05). Conclusion: Our results revealed that Tiagabine exhibits a potent neuroprotective effect against spinal cord I/R injury. The protection system operates through multiple channels instead of using a single defense system which includes three main mechanisms to reduce oxidative stress, control inflammation and prevent cell death. The reperfusion phase receives benefits from Tiagabine-induced GABAergic transmission because it counteracts glutamate-induced excitotoxicity. Our findings indicate that blocking GAT-1 could serve as a therapeutic approach to treat the types of injuries.

Anahtar Kelimeler

• Neuroprotection
• Tiagabine
• Spinal cord
• Ischemia/reperfusion injury

Kaynakça

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