Want Andrew, Nan Xinsheng, Kokkali Eirini, Barde Yves-Alain, Morgan James E
School of Optometry and Vision Sciences, Cardiff University, Cardiff CF24 4HQ, UK.
School of Bioscience, Cardiff University, Cardiff CF10 3AX, UK.
Brain Commun. 2023 Mar 2;5(2):fcad046. doi: 10.1093/braincomms/fcad046. eCollection 2023.
In humans and other primates, blood platelets contain high concentrations of brain-derived neurotrophic factor due to the expression of the gene in megakaryocytes. By contrast, mice, typically used to investigate the impact of CNS lesions, have no demonstrable levels of brain-derived neurotrophic factor in platelets, and their megakaryocytes do not transcribe significant levels of the gene. Here, we explore potential contributions of platelet brain-derived neurotrophic factor with two well-established CNS lesion models, using 'humanized' mice engineered to express the gene under the control of a megakaryocyte-specific promoter. Retinal explants prepared from mice containing brain-derived neurotrophic factor in platelets were labelled using DiOlistics and the dendritic integrity of retinal ganglion cells assessed after 3 days by Sholl analysis. The results were compared with retinas of wild-type animals and with wild-type explants supplemented with saturating concentrations of brain-derived neurotrophic factor or the tropomyosin kinase B antibody agonist, ZEB85. An optic nerve crush was also performed, and the dendrites of retinal ganglion cells similarly assessed 7-day post-injury, comparing the results of mice containing brain-derived neurotrophic factor in platelets with wild-type animals. In mice engineered to contain brain-derived neurotrophic factor in platelets, the mean serum brain-derived neurotrophic factor levels were 25.74 ± 11.36 ng/mL for homozygous and 17.02 ± 6.44 ng/mL for heterozygous mice, close to those determined in primates. Retinal explants from these animals showed robust preservation of dendrite complexity, similar to that seen with wild-type explants incubated with medium supplemented with brain-derived neurotrophic factor or the tropomyosin receptor kinase B antibody agonist, ZEB85. The Sholl areas under curve were 1811 ± 258, 1776 ± 435 and 1763 ± 256 versus 1406 ± 315 in the wild-type control group ( ≤ 0.001). Retinal ganglion cell survival based on cell counts was similar in all four groups, showing ∼15% loss. A robust neuroprotective effect was also observed following optic nerve crush when assessing the dendrites of the retinal ganglion cells in the transgenic mouse, with Sholl area under the curve significantly higher compared to wild-type (2667 ± 690 and 1921 ± 392, = 0.026), with no significant difference in the contralateral eye controls. Repeat experiments found no difference in cell survival, with both showing ∼50% loss. These results indicate that platelet brain-derived neurotrophic factor has a strong neuroprotective effect on the dendrite complexity of retinal ganglion cells in both an and model, suggesting that platelet brain-derived neurotrophic factor is likely to be a significant neuroprotective factor in primates.
在人类和其他灵长类动物中，由于巨核细胞中该基因的表达，血小板含有高浓度的脑源性神经营养因子。相比之下，通常用于研究中枢神经系统（CNS）损伤影响的小鼠，其血小板中没有可检测到的脑源性神经营养因子水平，并且它们的巨核细胞不会转录该基因的显著水平。在此，我们使用经过基因工程改造、在巨核细胞特异性启动子控制下表达该基因的“人源化”小鼠，通过两种成熟的CNS损伤模型来探索血小板脑源性神经营养因子的潜在作用。从小鼠制备的含有血小板脑源性神经营养因子的视网膜外植体用DiOlistics标记，并在3天后通过Sholl分析评估视网膜神经节细胞的树突完整性。将结果与野生型动物的视网膜以及补充了饱和浓度脑源性神经营养因子或原肌球蛋白激酶B抗体激动剂ZEB85的野生型外植体进行比较。还进行了视神经挤压，并在损伤后7天类似地评估视网膜神经节细胞的树突，将血小板中含有脑源性神经营养因子的小鼠结果与野生型动物进行比较。在经过基因工程改造使血小板中含有脑源性神经营养因子的小鼠中，纯合小鼠的平均血清脑源性神经营养因子水平为25.74±11.36 ng/mL，杂合小鼠为17.02±6.44 ng/mL，接近在灵长类动物中测定的水平。来自这些动物的视网膜外植体显示出树突复杂性的强大保存，类似于用补充了脑源性神经营养因子或原肌球蛋白受体激酶B抗体激动剂ZEB85的培养基孵育的野生型外植体。曲线下的Sholl面积分别为1811±258、1776±435和1763±256，而野生型对照组为1406±315（P≤0.001）。基于细胞计数的视网膜神经节细胞存活率在所有四组中相似，显示约15%的损失。在评估转基因小鼠视网膜神经节细胞的树突时，视神经挤压后也观察到了强大的神经保护作用，曲线下的Sholl面积显著高于野生型（2667±690和1921±392，P = 0.026），对侧眼对照无显著差异。重复实验发现细胞存活率无差异，两者均显示约50%的损失。这些结果表明，血小板脑源性神经营养因子在体外和体内模型中对视网膜神经节细胞的树突复杂性都有很强的神经保护作用，表明血小板脑源性神经营养因子可能是灵长类动物中一种重要的神经保护因子。
