Protection Of Spinal Cord Ischemia-reperfusion Injury By Means Of Adenosine Preconditioning In Rats

Establishment of spinal cord ischemia-reperfusion model in ratsObjective To establish spinal cord ischemia-reperfusion model in rats. Methods 15 male SD rats were used for preliminary experiment.15 male SD rats were used for formal experiment. The animals were anesthetized with chloral hydrate and fixed at supine position on the dissection station. To maintain body temperature at 38℃ during surgery with light. To dissect the anatomical position and tissue for surgery, expose the abdominal aorta. The experimental model was established in rats through cross-clamping of abdominal aorta 90 minutes from left renal artery to common iliac vein after tail vein injection with heparin. The animals were placed in cage after the intramuscular injection of penicillin. Spinal cord function was assessed by the Modified Tarlov Scale at 6,12,24h after reperfusion. At 24h after reperfusion, histologic analysis was done by HE staining. Results In preliminary experiment,9 survived and 6 died. In 9 cases the model successfully established, Tarlov score at 6h after reperfusion was 0.78±0.83, at 12h after reperfusion was 1.11±0.60, at 24h after reperfusion was 1.67±0.70.15 rats all survived in formal experiment. Tarlov score at 6h after reperfusion was 0.93±0.79, at 12h after reperfusion was 1.60±0.51, at 24h after reperfusion was 2.00±0.66.15 rats all survived in formal experiment. HE staining results showed:extensive spinal cord injury such as cell edema, lightly stained cytoplasm and chromatin margination was seen at 6h after reperfusion. HE staining showed neuron swelling, vacuoles formation in cytoplasm, unclear nuclear and wide cell gap at 12h after reperfusion. HE staining at 24h after reperfusion showed extensive spinal cord neurons and glial cells injury when compared to HE staining at 12h after reperfusion. Conclusions Successfully established spinal cord ischemia-reperfusion model in rats through cross-clamping of abdominal aorta 90 minutes.Effects of adenosine preconditioning on expressions of B cell leukemia/ lymphoma-2, bcl-2 associated X protein, GRP-78,caspase 12 following the spinal cord ischemia-reperfusion of ratsObjective Paraplegia is a serious complication after abdominal aorta surgery, the main reason is spinal cord ischemia-reperfusion injury, especially delayed neuronal death induced by abdominal aorta occlusion. In order to reduce this complication, we take different measure:controlling ischemic time, cooling ischemic spinal cord segments, draining cerebrospinal fluid, perfusing solution to tissue next to spinal cord and applying antioxidants or other drugs. As a protective agent in cardiac surgery, adenosine can decrease reperfusion injury in myocardium, but its ability to reduce postoperative spinal cord injury remains to be studied. To study the expressions of B cell leukemia/lymphoma-2 (bcl-2), bcl-2 associated X protein (bax), GRP-78, caspase 12 following ischemia-reperfusion injury and their action mechanism on apoptosis for investigating the protection of spinal cord I-R injury by means of adenosine preconditioning. Methods Twenty-seven male SD rats were randomly divided into three groups, group A(n= 9):sham group, group B(n= 9):ischemia-reperfusion group, and group C (n= 9):administration with adenosine group. The animals were anesthetized with chloral hydrate and fixed at supine position on the dissection station. To maintain body temperature at 38℃ during surgery with light. To dissect the anatomical position and tissue for surgery, expose the abdominal aorta. The experimental model was established in rats through cross-clamping of abdominal aorta 90 minutes from left renal artery to common iliac vein after tail vein injection with heparin. The animals were placed in cage after the intramuscular injection of penicillin. Spinal cord function was assessed by the Modified Tarlov Scale at 6,12,24h after reperfusion. At 24h after reperfusion, histologic analysis was done by HE staining, Nissl staining and TUNEL staining, expressions of bcl-2, bax, GRP-78, caspase 12 protein and mRNA were determined by Western blotting and RT-PCR respectively. Took L2-L5 spinal cord segments and placed in 4% paraformaldehyde for hematoxylin-eosin (HE) staining, Nissl staining and observed under light microscope. Took one paraffin section of the spinal cord specimens in each group, dewaxing, incubating with proteinase K and TUNEL reaction solution, Converter-POD solution. Stained by diaminobenzene hydrazine (DAB) and hematoxylin and observed under light microscop. Spinal cord segments were fixed in 2% paraformaldehyde-2.5% glutaraldehyde then transferred to 4℃ 1% osmium tetroxide solution. After 1 hour, it were gradient dehydrated by ethanol and acetone, embedded in epoxy resin, sliced into thin slides, double stained with uranyl acetate-lead citrate and observed ultrastructure of spinal cord neurons and glial cells under H-600 transmission electron microscope. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique and solid phase immunoassays were used in our experiment. Weighed 100mg spinal cord specimens using an electronic balance, cut into pieces, added Lysates and phenylmethylsulfonyl fluoride (PMSF), homogenized with a tissue homogenizer, and centrifuged at 4℃ to obtain supernatant, i.e. total protein. After determination of the concentration of total protein, using SDS-PAGE electrophoresis, then transferred to polyvinylidene fluoride (PVDF) membrane and blocked by blocking solution. Added diluted bcl-2 antibody (1:1000), bax antibody (1:4000), GRP-78 antibody (1:3000), caspase 12 antibody (1:1000) at 4℃ overnight,β- actin antibody as an internal reference. After washing the membrane, added goat anti-rabbit secondary antibody (1:5000) and incubated at room temperature for 1 h, then was colored by enhanced chemiluminescence (ECL) reagent. Using Gelpro32 analysis software analyzed absorbance and calculated the relative expressions. RT-qPCR:total RNA was extracted by Trizol and was reverse transcribed, adding primers of bcl-2, bax, GRP-78, caspase 12 and β-actin, cDNA synthesized was used for PCR amplification reaction. PCR conditions: 94℃ 3 min,94℃ 30 s,60℃ 30 s,72℃30 s,30 cycles of amplification,72℃ 10 min. Fluorescence quantitative PCR reaction system was 20.0μl and reaction conditions was 95℃ 10 min,95℃ 10 s,60℃ 20 s,72℃ 30 s,44 cycles. Melting curve analysis temperature was 72.0℃ to 94.0℃. PCR reaction was applied by Bio-Rad CFX96 real-time PCR instrument (American Bio-rad Co.) and amplified curve, standard curve, melting curve were drawn automatically by the instrument. Using Primer Premier 5.0 designed primers and sequence was as follows:bcl-2 primer sequences: upstream:5’-CGACTTTGCAGAGATGTCCA-3’, downstream:5’-ATGCCGGTTCAGGTACTC AG- 3’,223bp. bax primer sequences:upstream:5’ CTGCAGAGGATGATTGCTGA-3’downstream:5’-GATCAGCTCG GGCACTTTAG-3’,174bp. GRP-78 primer sequences:upstream:5’-TGACTCCAGCAGAGCCCTAT-3’, downstream:5’-TTGGGAGAACCATTGAGAGG-3’,197bp. caspase 12 primer sequences:upstream:5’-GCTGCCAAGAGAACACATGA-3’, downstream:5 ’-GGTTCTCAGCTTTGCTCAG G -3’,169bp. glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer sequences: upstream:5’-TGCCACTCAGAAGACTGTGG-3’,downstream:5’-TTCAGCTCTG GGATGACCTT-3’,129bp. Calculated bcl-2, bax, GRP-78, caspase 12 mRNA expression using GAPDH as an internal reference. Results We can see large neuron body, rich cytoplasm, no bleeding or edema, no inflammatory cell infiltration, normal Nissl body in group A. HE staining and NISSL staining showed neuron swelling, vacuoles formation in cytoplasm, unclear nuclear, wide cell gap and unclear Nissl body in group B. Group C showed neuron swelling, clear nuclear, wide cell gap, but damage was significantly reduced compared with group B. Electron microscopy in group A showed: intact nuclear membrane, well distributed chromatin, normal perinuclear organelles, neatly arranged myelin, normal axon. We can see nucleus shrinkage, chromatin margination, loose myelin, voids between axon and myelin in group B. Group C showed intact nuclear membrane, well distributed chromatin, intact but a little loose myelin, normal axon and little vacuoles in matrix. TUNEL staining showed:well stained neurons and rare positive cells in group A. Positive cells contained many purple and blue granules were less in group C than in group B. Western blot analysis showed:Bcl-2 protein expressions were the least in group B, protein expression levels in group C were higher than group B. bax protein expressions were the highest in group B, protein expression levels in group C were lower compared with group B. Bcl-2/bax expression levels in group C were higher than group B, the difference was statistically significant (P<0.01). Bcl-2/bax expression level in group A was higher than group B and group C, the difference was statistically significant (P<0.01). GRP-78 protein expressions in group C were higher than group B, the difference was statistically significant (P<0.01). Caspase 12 protein expressions in group A were higher than group B and group C, the difference was statistically significant (P <0.01). Caspase 12 protein expressions in group C were lower than group B, the difference was statistically significant (P<0.01). RT-qPCR showed:Bcl-2 expressions were the least in group B, bcl-2 expressions were higher in group C than group B. Bax expressions were the strongest in group B, bax expressions were higher in group B compared with group C. Bcl-2/bax expression levels in group C were higher than group B, the difference was statistically significant (P<0.01). Bcl-2/bax expression levels in Group A were higher than group B and group C, the difference was statistically significant (P<0.01). GRP-78 expression levels were the weakest in group B, GRP-78 expression levels in group C were higher than group B, the difference was statistically significant (P<0.01). Caspase 12 expression levels were the strongest in group B, caspase 12 expression levels in group B were higher than group C, the difference was statistically significant (P<0.01).Conclisions At 24 h after reperfusion, apoptotic cells of spinal cord in administration with adenosine group were less than ischemia-reperfusion group, adenosine can inhibit cell apoptosis. Bcl-2/bax expression levels in administration with adenosine group were higher than ischemia-reperfusion group. Adenosine can increase the expression of bcl-2 and reduce the expression of bax, thereby inhibiting apoptosis. Adenosine can also raise the expression of GRP-78 and decrease the expression of caspase 12, inhibiting endoplasmic reticulum stress, further inhibiting apoptosis of spinal cord neurons and glial cells.