Can Cytoprotective Cobalt Protoporphyrin Protect Skeletal Muscle and Muscle-derived Stem Cells From Ischemic Injury?

Background

Extremity trauma is the most common injury seen in combat hospitals as well as in civilian trauma centers. Major skeletal muscle injuries that are complicated by ischemia often result in substantial muscle loss, residual disability, or even amputation, yet few treatment options are available. A therapy that would increase skeletal muscle tolerance to hypoxic damage could reduce acute myocyte loss and enhance preservation of muscle mass in these situations.

Questions/purposes

In these experiments, we investigated (1) whether cobalt protoporphyrin (CoPP), a pharmacologic inducer of cytoprotective heme oxygenase-1 (HO-1), would upregulate HO-1 expression and activity in skeletal muscle, tested in muscle-derived stem cells (MDSCs); and (2) whether CoPP exposure would protect MDSCs from cell death during in vitro hypoxia/reoxygenation. Then, using an in vivo mouse model of hindlimb ischemia/reperfusion injury, we examined (3) whether CoPP pharmacotherapy would reduce skeletal muscle damage when delivered after injury; and (4) whether it would alter the host inflammatory response to injury.

Methods

MDSCs were exposed in vitro to a single dose of 25 μΜ CoPP and harvested over 24 to 96 hours, assessing HO-1 protein expression by Western blot densitometry and HO-1 enzyme activity by cGMP levels. To generate hypoxia/reoxygenation stress, MDSCs were treated in vitro with phosphate-buffered saline (vehicle), CoPP, or CoPP plus an HO-1 inhibitor, tin protoporphyrin (SnPP), and then subjected to 5 hours of hypoxia (< 0.5% O₂) followed by 24 hours of reoxygenation and evaluated for apoptosis. In vivo, hindlimb ischemia/reperfusion injury was produced in mice by unilateral 2-hour tourniquet application followed by 24 hours of reperfusion. In three postinjury treatment groups (n = 7 mice/group), CoPP was administered intraperitoneally during ischemia, at the onset of reperfusion, or 1 hour later. Two control groups of mice with the same injury received phosphate-buffered saline (vehicle) or the HO-1 inhibitor, SnPP. Myocyte damage in the gastrocnemius and tibialis anterior muscles was determined by uptake of intraperitoneally delivered Evans blue dye (EBD), quantified by image analysis. On serial sections, inflammation was gauged by the mean myeloperoxidase staining intensity per unit area over the entirety of each muscle.

Results

In MDSCs, a single exposure to CoPP increased HO-1 protein expression and enzyme activity, both of which were sustained for 96 hours. CoPP treatment of MDSCs reduced apoptotic cell populations by 55% after in vitro hypoxia/reoxygenation injury (from a mean of 57.3% apoptotic cells in vehicle-treated controls to 25.7% in CoPP-treated cells, mean difference 31.6%; confidence interval [CI], 28.1–35.0; p < 0.001). In the hindlimb ischemia/reperfusion model, CoPP delivered during ischemia produced a 38% reduction in myocyte damage in the gastrocnemius muscle (from 86.4% ± 7% EBD⁺ myofibers in vehicle-treated, injured controls to 53.2% EBD⁺ in CoPP-treated muscle, mean difference 33.2%; 95% CI, 18.3, 48.4; p < 0.001). A 30% reduction in injury to the gastrocnemius was seen with drug delivery at the onset of reperfusion (to 60.6% ± 13% EBD⁺ with CoPP treatment, mean difference 25.8%; CI, 12.2–39.4; p < 0.001). In the tibialis anterior, however, myocyte damage was decreased only when CoPP was given at the onset of reperfusion, resulting in a 27% reduction in injury (from 78.8% ± 8% EBD⁺ myofibers in injured controls to 58.3% ± 14% with CoPP treatment, mean difference 20.5%; CI, 6.1–35.0; p = 0.004). Delaying CoPP delivery until 1 hour after tourniquet release obviated the protective effect in both muscles. Mean MPO staining intensity per unit area, indicating the host inflammatory response, decreased by 27–34% across both the gastrocnemius and tibialis anterior muscles when CoPP was given either during ischemia or at the time of reperfusion. Delaying drug delivery until 1 hour after the start of reperfusion abrogated this antiinflammatory effect.

Conclusions

CoPP can decrease skeletal muscle damage when given early in the course of ischemia/reperfusion injury and also provide protection for regenerative stem cell populations.

Clinical Relevance

Pharmacotherapy with HO-1 inducers, delivered in the field, on hospital arrival, or during trauma surgery, may improve preservation of muscle mass and muscle-inherent stem cells after severe ischemic limb injury.

Electronic supplementary material

The online version of this article (doi:10.1007/s11999-015-4332-8) contains supplementary material, which is available to authorized users.     

偏心髋臼旋转截骨术的生物力学机制及初步临床疗效

 目的 探讨偏心髋臼旋转截骨术治疗髋关节发育不良的生物力学机制及其初步临床疗效。方法 取6具经福尔马林防腐处理的女性尸体骨盆标本,建立髋关节生物力学模型,在模型上模拟偏心髋臼旋转截骨术。对骨盆缓慢施加连续纵向压力0~500 N,测量术前和术后载荷100、200、300、400、500 N时的股骨头承重区应变值,计算应力值。2007年7月至2014年10月应用偏心髋臼旋转截骨术治疗髋关节发育不良25例（26髋）,男6例,女19例;年龄11~57岁,平均31岁。术后以Harris髋关节评分评价髋关节功能,摄骨盆正位X线片测量头臼指数、中心边缘角（center-edge-angle,CE角）及Sharp角。结果-随着脊柱纵向压力加大,股骨头上的应力值随之增加。偏心髋臼旋转截骨术后应力值在载荷超过300 N后由上升趋势转变为下降趋势,总体呈抛物线状。100~500 N载荷下偏心髋臼旋转截骨术后的应力值与术前差异均无统计学意义。临床随访18例（19髋）,随访率72%。随访时间7~85个月,平均40个月。Harris髋关节评分由术前（64.3±7.2）分提高至末次随访时（85.6±5.3）分;头臼指数平均增加36.5%、CE角平均增加33.1°、Sharp角平均减少12.3°,与术前比较差异均有统计学意义。结论-偏心髋臼旋转截骨术具有较好的矫正髋臼畸形的能力,可增大股骨头的髋臼覆盖面和降低承重区压力。     

Human umbilical cord blood stem cells and brain-derived neurotrophic factor for optic nerve injury:a biomechanical evaluation

Treatment for optic nerve injury by brain-derived neurotrophic factor or the transplantation of human umbilical cord blood stem cells has gained progress, but analysis by biomechanical indicators is rare. Rabbit models of optic nerve injury were established by a clamp. At 7 days after injury, the vitreous body received a one-time injection of 50 μg brain-derived neurotrophic factor or 1 × 106 human umbilical cord blood stem cells. After 30 days, the maximum load, maximum stress, maximum strain, elastic limit load, elastic limit stress, and elastic limit strain had clearly improved in rabbit models of optical nerve injury after treatment with brain-derived neurotrophic factor or human umbilical cord blood stem cells. The damage to the ultrastructure of the optic nerve had also been reduced. These findings suggest that human umbilical cord blood stem cells and brain-derived neurotrophic factor effectively repair the injured optical nerve, improve biomechanical properties, and contribute to the recovery after injury.     

How disgust facilitates avoidance: an ERP study on attention modulation by threats

This study investigated the attention modulation of disgust in comparison with anger in a dot-probe task. Results indicated a two-stage processing of attention modulation by threats. When participants viewed the cues that were represented by Chinese faces (i.e. the in-group condition), it was found at the early processing stage that an angry face elicited a larger occipital P1 component whereas a disgusted face elicited a smaller P1 for validly than for invalidly cued targets. However, the result pattern was reversed at the later processing stage: the P3 amplitudes were larger for valid disgust cues but were smaller for valid angry cues, when both were compared with invalid cue conditions. In addition, when participants viewed the cues that were represented by foreign faces (i.e. the out-group condition), the attention modulation of disgust/anger diminished at the early stage, whereas enhanced P3 amplitudes were observed in response to validly cued targets in both disgusting and angry conditions at the later stage. The current result implied that although people can perceptually differentiate the emotional categories of out-group faces as accurately as in-group faces, they may still be not able to psychologically understand the subtle differences behind different categories of out-group facial expressions.