Treatment with Gallium Maltolate after spinal cord thoracic contusion in female mice, functional outcome and inflammatory markers

STUDY PURPOSE: Hemorrhage after SCI is associated with worse functional outcome, compared to non-hemorrhage SCI cases. Toxicity and pro-inflammatory effects of hemoglobin, its breakdown factors, and iron deposition may contribute to this problem. Gallium nitrate and Gallium maltolate are used in cancer therapy and in treatment of bacterial infections as iron competitors, resulting in iron starvation and cell death of bacteria or cancer cells, which have a high iron demand. While this mechanism is not necessarily relevant to control tissue hemorrhage after SCI, the additional anti-inflammatory role of gallium maltolate made it an interesting treatment consideration. DATA COLLECTED: This experiment was performed in two cohorts of female, 8-week-old C57BL/6 mice (Charles River Laboratories). Under isoflurane anesthesia, mice were subjected to a 40 kdyne contusion (Infinite Horizon Impactor) injury at T11. In the first cohort, gallium maltolate versus a saline control (n=8-10) was injected subcutaneously immediately after injury, as well as at two and at four days later (200mg/KG). In contrast to earlier reports, this dose was not well tolerated: some mice died and the experiment was terminated at day 7. In the second cohort, mice received 25mg/KG gallium maltolate or saline s.c. on day 0 (immediately after injury), and every second day for the survival period (three or seven days). This dose was well tolerated. At day 3 or 7 after SCI, mice were euthanized with an overdose of Phenytoin/ Pentobarbital (120 mg/kg) and transcardially perfused with ice cold 1x PBS. A 5 mm piece of spinal cord centering on the lesion was dissected out, and immediately snap frozen. RNA was extracted using the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, followed by RNA quantification and characterization of purity using Nanodrop (Thermo Scientific) and reverse transcription of 1 µg of RNA using QuantiTect Reverse Transcription Kit (Qiagen). Q-PCR was performed in duplicates using a PCR thermal cycler (LightCycler® 480 System, Roche) with specific primers using LightCycler® 480 Mastermix (Roche). Gene expression levels were analyzed using the ddct method normalized to peptidylprolyl isomerase A (PPIA) as a housekeeping gene and laminectomy as a baseline. The following primers were used for Q-PCR: PPIA: ATG TGC CAG GGT GGT GAC TTT A (forward primer 5'–3'); TGT GTT TGG TCC AGC ATT TGC C (reverse primer 5'–3'), TNF: TTG CTC TGT GAA GGG AAT GG (forward primer 5'–3'); GGC TCT GAG GAG TAG ACA ATA AAG (reverse primer 5'–3'), IL-1b: ATG GGC AAC CAC TTA CCT ATT T (forward primer 5'–3'); GTT CTA GAG AGT GCT GCC TAA TG (reverse primer 5'–3'), IL-6: CTT CCA TCC AGT TGC CTT CT (forward primer 5'–3'); CTC CGA CTT GTG AAG TGG TAT AG (reverse primer 5'–3'). iNOS: GAA CGG AGA ACG TTG GAT TTG (forward primer 5'–3'); TCA GGT CAC TTT GGT AGG ATT T (reverse primer 5'–3'), bax: CAT CTT CTT CCA GAT GGT GA (forward primer 5'–3'); GTT TCA TCC AGG ATC GAG CAG (reverse primer 5'–3'). DATA USAGE NOTES: Cohort 1: The average BMS score was significantly lower in gallium-treated mice at day 1 and 3. A lower number of gallium-treated mice were able to support their weight at day 1, 3 and 5 after SCI. Cohort 2: BMS scores and weight support at day 1 after injury were not different between groups. Q-PCR revealed no differences in expression levels of TNF, IL-6, IL-1ß, bax or iNOS between gallium maltolate and saline treated mice at day 3 and 7 after SCI. Bax, IL6 and iNOS showed a trend for higher expression levels in gallium-treated mice at day 3 after injury. Gallium treatment did not improve the functional outcome after SCI early after injury. However, the deterioration compared to controls could also have been caused by worse overall physiologic conditions of the mice. In the second cohort, no difference was seen between the groups at day 1. No anti-inflammatory effect was detected by Q-PCR at day 3 or 7. In summary, we did not detect any beneficial effect on functional outcome or Q-PCR level in gallium maltolate-treated mice.研究目的：与非出血性脊髓损伤（Spinal Cord Injury, SCI）病例相比，脊髓损伤后出血患者的功能预后更差。血红蛋白、其降解产物以及铁沉积的毒性与促炎效应，可能是导致该不良预后的原因。硝酸镓（Gallium nitrate）和麦芽糖酸镓（Gallium maltolate）作为铁竞争剂，用于癌症治疗与细菌感染治疗，通过造成铁饥饿来诱导高铁需求的细菌或癌细胞死亡。尽管该机制未必适用于调控脊髓损伤后的组织出血，但麦芽糖酸镓额外的抗炎特性使其成为颇具研究价值的治疗候选方案。数据采集：本实验使用两批雌性8周龄C57BL/6小鼠（查尔斯河实验室（Charles River Laboratories））开展。小鼠在异氟烷（Isoflurane）麻醉下，于T11节段接受40千达因（kdyne）的脊髓挫伤造模（使用Infinite Horizon脊髓打击器）。第一批次小鼠于造模后即刻、术后第2天及第4天皮下注射200mg/kg的麦芽糖酸镓或生理盐水对照（每组n=8-10）。与既往研究报道不同，该剂量耐受性不佳：部分小鼠死亡，实验于第7天终止。第二批次小鼠于造模后即刻（第0天）开始，以25mg/kg的剂量皮下注射麦芽糖酸镓或生理盐水，每2天给药1次，存活期设为3天或7天。该剂量耐受性良好。于脊髓损伤后第3天或第7天，以过量苯妥英钠/戊巴比妥（120mg/kg）处死小鼠，经心脏灌注冰冷的1×磷酸盐缓冲液（Phosphate Buffered Saline, PBS）。取以损伤灶为中心的5mm长脊髓组织，立即置于液氮中快速冷冻。按照试剂盒说明书，使用RNeasy脂质组织微量试剂盒（RNeasy Lipid Tissue Mini Kit，凯杰（Qiagen），德国希尔登）提取RNA；随后采用Nanodrop分光光度计（Thermo Scientific）对RNA进行定量与纯度检测，并使用QuantiTect反转录试剂盒（QuantiTect Reverse Transcription Kit，凯杰）对1μg RNA进行反转录。采用LightCycler® 480实时荧光定量PCR系统（LightCycler® 480 System，罗氏（Roche））及LightCycler® 480预混反应液（罗氏），以特异性引物进行重复孔定量聚合酶链反应（Quantitative Polymerase Chain Reaction, Q-PCR）。以肽基脯氨酰异构酶A（peptidylprolyl isomerase A, PPIA）作为内参基因，以椎板切除术组作为基线，采用ΔΔCt法分析基因表达水平。本实验Q-PCR所用引物如下：PPIA：正向引物5'→3'：ATG TGC CAG GGT GGT GAC TTT A；反向引物5'→3'：TGT GTT TGG TCC AGC ATT TGC C；肿瘤坏死因子α（Tumor Necrosis Factor-α, TNF）：正向引物5'→3'：TTG CTC TGT GAA GGG AAT GG；反向引物5'→3'：GGC TCT GAG GAG TAG ACA ATA AAG；白细胞介素1β（Interleukin-1β, IL-1β）：正向引物5'→3'：ATG GGC AAC CAC TTA CCT ATT T；反向引物5'→3'：GTT CTA GAG AGT GCT GCC TAA TG；白细胞介素6（Interleukin-6, IL-6）：正向引物5'→3'：CTT CCA TCC AGT TGC CTT CT；反向引物5'→3'：CTC CGA CTT GTG AAG TGG TAT AG；诱导型一氧化氮合酶（Inducible Nitric Oxide Synthase, iNOS）：正向引物5'→3'：GAA CGG AGA ACG TTG GAT TTG；反向引物5'→3'：TCA GGT CAC TTT GGT AGG ATT T；Bcl-2相关X蛋白（Bcl-2 Associated X Protein, Bax）：正向引物5'→3'：CAT CTT CTT CCA GAT GGT GA；反向引物5'→3'：GTT TCA TCC AGG ATC GAG CAG。数据使用说明：第一批次：镓治疗组小鼠在造模后第1天和第3天的巴索小鼠评分（Basso Mouse Scale, BMS）显著低于对照组；造模后第1、3、5天，能够支撑体重的镓治疗组小鼠数量更少。第二批次：两组小鼠造模后第1天的巴索小鼠评分与体重支撑能力无显著差异。定量PCR结果显示，脊髓损伤后第3天和第7天，麦芽糖酸镓治疗组与生理盐水对照组小鼠的TNF、IL-6、IL-1β、Bax及iNOS基因表达水平无显著差异。造模后第3天，镓治疗组小鼠的Bax、IL-6及iNOS基因表达水平呈升高趋势。早期脊髓损伤后，镓治疗未能改善小鼠的功能预后。但与对照组相比的功能恶化，也可能源于小鼠整体生理状态较差。第二批次实验中，两组小鼠在造模后第1天无显著差异；定量PCR结果未在造模后第3天或第7天检测到抗炎效应。综上，本研究未在麦芽糖酸镓治疗组小鼠中观察到对功能预后或基因表达水平的有益影响。