Ludwig Boltzmann Institute for Traumatology, The Research Center in Cooperation with AUVA, 1200 Vienna, Austria.
Austrian Cluster for Tissue Regeneration, 1200 Vienna, Austria.
Biomolecules. 2024 Apr 30;14(5):543. doi: 10.3390/biom14050543.
Limited substrate availability because of the blood-brain barrier (BBB) has made the brain develop specific molecular mechanisms to survive, using lactate synthesized by astrocytes as a source of energy in neurons. To understand if lactate improves cellular viability and susceptibility to glutamate toxicity, primary cortical cells were incubated in glucose- or lactate-containing media and toxic concentrations of glutamate for 24 h. Cell death was determined by immunostaining and lactate dehydrogenase (LDH) release. Mitochondrial membrane potential and nitric oxide (NO) levels were measured using Tetramethylrhodamine, methyl ester (TMRM) and 4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate (DAF-FM) live staining, respectively. LDH activity was quantified in single cells in the presence of lactate (LDH substrate) and oxamate (LDH inhibitor). Nuclei of cells were stained with DAPI and neurons with MAP2. Based on the distance between neurons and glial cells, they were classified as linked (<10 µm) and non-linked (>10 µm) neurons. Lactate increased cell death rate and the mean value of endogenous NO levels compared to glucose incubations. Mitochondrial membrane potential was lower in the cells cultured with lactate, but this effect was reversed when glutamate was added to the lactate medium. LDH activity was higher in linked neurons compared to non-linked neurons, supporting the hypothesis of the existence of the lactate shuttle between astrocytes and at least a portion of neurons. In conclusion, glucose or lactate can equally preserve primary cortical neurons, but those neurons having a low level of LDH activity and incubated with lactate cannot cover high energetic demand solely with lactate and become more susceptible to glutamate toxicity.
由于血脑屏障 (BBB) 的存在，导致大脑发展出特定的分子机制来利用星形胶质细胞合成的乳酸作为神经元的能量来源，从而在有限的底物供应下存活。为了了解乳酸是否能提高细胞活力和对谷氨酸毒性的敏感性，将原代皮质细胞在含有葡萄糖或乳酸的培养基和有毒浓度的谷氨酸中孵育 24 小时。通过免疫染色和乳酸脱氢酶 (LDH) 释放来确定细胞死亡。使用 Tetramethylrhodamine, methyl ester (TMRM) 和 4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate (DAF-FM) 活体染色分别测量线粒体膜电位和一氧化氮 (NO) 水平。在存在乳酸 (LDH 底物) 和噁唑酸盐 (LDH 抑制剂) 的情况下，在单个细胞中定量测定 LDH 活性。用 DAPI 染色细胞核，用 MAP2 染色神经元。根据神经元和神经胶质细胞之间的距离，将它们分为相连（<10 µm）和不相连（>10 µm）神经元。与葡萄糖孵育相比，乳酸增加了细胞死亡率和内源性 NO 水平的平均值。在含有乳酸的细胞中，线粒体膜电位较低，但当将谷氨酸添加到乳酸培养基中时，这种效应被逆转。与不相连的神经元相比，相连的神经元中的 LDH 活性更高，这支持了星形胶质细胞和至少一部分神经元之间存在乳酸穿梭的假说。总之，葡萄糖或乳酸都可以同样地保存原代皮质神经元，但那些 LDH 活性低且用乳酸孵育的神经元不能仅用乳酸来满足高能量需求，并且更容易受到谷氨酸毒性的影响。
