To the Editor: Traumatic brain injury (TBI) refers to brain tissue damage caused by trauma. TBI pathogenesis is complicated and the involved molecular targets are also not clear. Astrocytes proliferate and hypertrophy after brain injury. They have strong tolerance to hypoxia and ischemia to protect neurons in the acute phase of brain injury. Exosomes, as small vesicles to exchange material and transmit information between cells, have been reported to exhibit potentials against various impairments in different disease models, including neurological diseases. One recent study has shown that exosomes are closely related to trauma, which may decrease A1 astrocytes by down-regulation of phosphorylation of nuclear factor-kappa B (NF-κB) in spinal cord injury. Our previous study demonstrated that the TBI could promote exosome release, suggesting the capability of self-protection by exosomes after impairment. Connexin 43 (Cx43, also called gap junction alpha 1 [GJA1]) is an important gap junction protein with widest expression.
GJA1-20k is a non-classical Cx43 protein with a fully truncated N-terminal. Our previous study showed that GJA1-20k overexpression in astrocytes promoted the recovery and viability of neurons in TBI by regulating mitochondrial function. Herein, the present study aimed to explore the protective effects of exosomes from GJA1-20k overexpressed astrocytes on TBI.
Exosomes were isolated from astrocytes transducted with lentiviral vector GJA1-20k, defined as exosome-LV-GJA1-20k. Exosome-LV-GJA1-20k were administrated to TBI rats by tail vein injection. Morris Water maze test and modified neurological severity score were employed to detect cognitive impairment and neurological deficits. Cell apoptosis and autophagy were examined in the rats treated with exosomes. The detailed experimental procedures can be found in the Supplementary methods, https://links.lww.com/CM9/B301.
The transduction efficacy of GJA1-20k into primary astrocytes was firstly examined. From Supplementary Figure 1A, https://links.lww.com/CM9/B301, it could be clearly seen that lentiviral vector successfully transferred GJA1-20k to primary astrocytes. GJA1-20k expression increased nearly three times compared to the control group. In astrocyte-derived exosomes, the expression of GJA1-20k was also higher in GJA1-20k transducted astrocytes-derived exosomes than that in control group [Supplementary Figure 1B, https://links.lww.com/CM9/B301]. These results showed that GJA1-20k transduction not only upregulated the expression of GJA1-20k in primary astrocytes, but also upregulated the expression of GJA1-20k protein in isolated exosomes.
In order to explore the effects of exosome-LV-GJA1-20k on neurological deficits and brain edema caused by TBI, the neurological deficit scores were measured at pre-operation, day 3, 7, and 14 post-TBI; and brain water content was detected at day 3 post-TBI. After TBI, treatment with the exosomes isolated from astrocytes without transduction of GJA1-20k had no effects on TBI rats, while exosome-LV-GJA1-20k significantly improved the neurological deficits on day 3, 7, and 14 [Figure 1A] and decreased brain edema [Figure 1B]. It also showed that exosome-LV-GJA1-20k had no effect on normal rats. Therefore, in the following study, we only selected the normal control group (sham group), the TBI group, and the TBI group subjected with exosome-LV-GJA1-20k treatment.
In order to explore the effects of exosome-LV-GJA1-20k on cognitive impairment in TBI rats, Morris water maze assay was applied during day 19–day 22 after TBI. In 3 days of training sessions, the escape latencies decreased gradually in each group, but the TBI group had significantly higher latency than the sham group and the treatment of exosome-LV-GJA1-20k perceptibly decreased the escape latency of TBI rats on day 20 and 21 [Figure 1C]. The average swim speed of the TBI group was lower than that of the sham group, while exosome-LV-GJA1-20k treatment increased the average speed of TBI rats [Figure 1D]. In the probe trial, TBI group rats spent less time in the target quadrant than the sham group rats, and the administration of exosome-LV-GJA1-20k extended the time in the target quadrant of TBI rats [Figure 1E]. When removing the platform, the number of platform site crossings were recorded. The results showed that TBI group rats had less numbers in crossing the non-existent platform compared to the control group, while exosome-LV-GJA1-20k treatment increased the number of platform site crossings, indicating an improvement in cognitive impairments [Figure 1F].
To further detect the effects of exosome-LV-GJA1-20k on the TBI-induced cell apoptosis, qRT-PCR was used to analyze the mRNA levels of Bax and Bcl-2, while Western blotting was used to analyze the protein levels of cleaved caspase-3, Bax, and Bcl-2 in the lesioned cortices’ tissues of experimental rats on day 7 post-injury. It was further revealed that exosome-LV-GJA1-20k administration decreased Bax and increased Bcl-2 expression both in mRNA and protein levels of TBI rats, together with a decrease of cleaved caspase-3 expression in protein level [Figure 1G-L]. These data indicated that the exosome-LV-GJA1-20k inhibited cell apoptosis in the lesioned cortices’ tissues induced by TBI in rats.
In order to illustrate the potential of exosome-LV-GJA1-20k on autophagy, Western blotting was used to analyze the proteins levels of light chain 3 (LC3) I/II, Beclin-1, antithymocyte globulin 3 (ATG-3), and ATG-7 in the lesioned cortices’ tissues of experimental rats at day 7 post-injury. The proteins levels of LC3 I/II, Beclin-1, ATG-3, and ATG-7 increased after TBI and exosome-LV-GJA1-20k treatment further boosted the increase [Supplementary Figure 2A-E, https://links.lww.com/CM9/B301], indicating that exosome-LV-GJA1-20k promoted autophagy in the lesioned cortices’ tissues of experimental TBI rats.
Overall, our study showed that transduction of GJA1-20k into primary astrocytes upregulated the expressions of GJA1-20k in both primary astrocytes and astrocyte-derived exosomes. The administration of exosome-LV-GJA1-20k not only significantly improved the neurological deficits and decreased brain edema but also ameliorated cognitive impairment induced by TBI in rats. In addition, exosome-LV-GJA1-20k treatment inhibited cell apoptosis and promoted mitochondrial autophagy in the rat lesioned cortices’ tissues. In summary, exosome-LV-GJA1-20k may promote mitochondrial autophagy and ameliorate apoptosis in TBI, further improving the cognitive damage in TBI rats.
The study was supported by grants from the Outstanding Youth Project of Jiangxi Province (No. 2020ACBL216005), General Program of National Natural Science Foundation of China (Nos. 82171366, 81960236), Key Project of Jiangxi Provincial Department of Education (No. GJJ190022), Project of Jiangxi Provincial Department of Health Commission (No. 20193199), Key Research and Development of Science at Technology Department of Jiangxi Province (Nos. 20203BBGL73172, 20192BBGL70022), and Shanghai Natural Science Foundation (No. 19ZR1447400).
Conflicts of interest
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