CXCL1 is predominantly expressed in cerebral cortex astrocytes after TBI
To visualize the cellular localization of CXCL1 in the cortex, we performed immunofluorescence double staining of CXCL1 with the astrocyte marker GFAP, the neuronal marker NeuN, and the microglia marker IBA-1, respectively. As shown in Figure 1, about 69% of CXCL1 was co-labeled with GFAP, about 30% with NeuN, and about 1% with IBA-1. The results suggested that CXCL1 was mainly expressed in astrocytes in the cortex of the injured area after TBI.
Figure 1. CXCL1 is expressed in astrocytes in the cortex of the injured area after TBI. CXCL1 is co-labeled with astrocyte marker GFAP (A-C). Some co-localization is visible with neuronal marker NeuN (D-F), and to a lesser extent the microglial marker IBA-1 (G-I). Co-labeling rate of CXCL1 with astrocytes is about 69%, with neurons 30%, and with microglia about 1% (J) (Bar=20 μm).
CXCR2 is mainly expressed in cerebral cortex neurons after TBI
To confirm the cellular localization of CXCR2 in the cortex, we employed immunofluorescence double staining of CXCR2 with the cellular markers GFAP, NeuN, and IBA-1, respectively. Figure 2 shows that about 85% of CXCR2 was co-labeled with the neuronal marker NeuN, 13% with the microglial marker IBA-1, and 2% with the astrocyte marker GFAP. The results indicated that CXCR2 was mainly expressed in neurons in the cortex of the injured area after TBI.
Figure 2. CXCR2 is expressed in neurons in the cortex of the injured area after TBI. CXCR2 is co-labeled with neuronal marker NeuN (D-F). Minimal co-labeling is visible with astrocyte marker GFAP (A-C) and microglial marker IBA-1 (G-I). Co-labeling rate of CXCR2 with astrocytes is about 2%, with neurons about 85%, and with microglia about 13% (J) (Bar=20 μm).
Upregulated CXCL1 and CXCR2 mRNA and protein expression in rat cerebral cortex after TBI
The expression level of CXCL1 and CXCR2 mRNA and protein were measured by RT-qPCR and ELISA, respectively, in the peri-injured cortex at 1, 3, 7, and 10 days after TBI. As shown in Figure 3A, the mRNA expression levels of CXCL1 and CXCR2 peaked on the first day and then decreased compared with the sham group. The protein expression of inflammation-related factors CXCL1 and CXCR2, as shown in Figure 3B, peaked on the first and third day, respectively, and then showed a decreasing trend compared with the sham group.
Figure 3. mRNA and protein expression of CXCL1 and CXCR2 are upregulated after TBI. (A) Compared to sham, mRNA expression of CXCL1 and CXCR2 after TBI peaks on the first day and then decreases. All values are expressed as mean ± SEM (n=6). (B) Compared to sham, expression of CXCL1 and CXCR2 protein after TBI peaks on the first and third day, respectively, and then decreases. ***p <0.001, *p <0.05.
Up-regulated p-ERK, p-JNK, and p-NF-κB is upregulatedexpression in rat cerebral cortex after TBI
The expression trends of p-ERK, p-JNK, and p-NF-κB in the cortex of the injured area were assessed by western Blot at 1, 3, 7, and 10 days after TBI. As shown in Figure 4, compared with the sham group, p-ERK and p-JNK peaked on the third day after TBI, whereas p-NF-κB showed a decreasing trend after peaking on the first day after TBI.
Figure 4. Up-regulation of p-ERK, p-JNK, and p-NF-κB after TBI. p-ERK and p-JNK expression peaks on the third day after TBI, and p-NF-κB is decreased after peaking on the first day after TBI. Values are expressed as mean ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05, vs. sham.
Increased apoptosis of cortical neurons in the injured area after TBI in rats
TUNEL staining was employed to detect apoptosis at 1, 3, and 7 days after TBI. Figure 5 shows that the number of TUNEL-positive cells increased at 1, 3, and 7 days after TBI compared with the sham group and showed a decreasing trend after reaching a peak on the first day.
Figure 5. Increased apoptosis of neuronal cells in the damaged cortex after TBI. (A-C) TUNEL-positive cells (green), DAPI (blue) and TUNEL+ DAPI merge. (D-G) Apoptosis-positive cells in the injured cortex at 1, 3, and 7 d after TBI. (H) TUNEL-positive cells decrease after peaking on the first day. Values are expressed as mean ± SEM. *** p <0.001, *p <0.05, vs. sham (Bar=20 μm).
CXCR2 antagonist improves neurological function in TBI Rats
To evaluate the effect of CXCL1-CXCR2 on the neurological function of TBI rats, mNSS was scored after cortical injection of CXCR2 antagonist in the injured area of TBI rats. As shown in Figure 6, the mNSS of rats after TBI was significantly higher than that of the sham group with impaired neurological function. The mNSS score of the TBI +high dose group was lower than that of the TBI+vehicle group after successive 3 days local injection of CXCR2 antagonist in the brain injury area, indicating that down-regulation of CXCR2 expression improves neurological function.
Figure 6. Improvement of neurological function in TBI rats after application of high dose of CXCR2 antagonist. mNSS scores decrease in TBI rats after application of high-dose CXCR2 antagonist SB225002. Values are expressed as mean ± SEM. ***p <0.001, vs. TBI vehicle; ###p <0.001, vs. sham.
CXCR2 antagonist improves cognitive function in TBI Rats
To evaluate the effect of CXCL1-CXCR2 on the cognitive function of TBI rats, the number of platform crossings and the average escape latency were observed after cortical injection of CXCR2 antagonist in the injured area of TBI rats. As shown in Figure 7, compared with the sham group, the escape latency of TBI rats was significantly prolonged and the number of platform crossings was reduced. The escape latency of the TBI +high dose group was lower than that of the TBI + vehicle group, while the number of platform crossings was higher.
Figure 7. Cognitive function of TBI rats is improved after application of high-dose CXCR2 antagonist SB225002. (A) The escape latency in TBI rats is shortened after high dose of CXCR2 antagonist. (B) The number of platform crossings in TBI rats is increased after high dose of CXCR2 antagonist. Values are expressed as mean ± SEM. **p < 0.01, *p < 0.05, vs. TBI vehicle; ###p < 0.001, vs. sham.
CXCR2 antagonist inhibits neuronal apoptosis in TBI rats.
To evaluate the effect of CXCR2 antagonist on the apoptosis of cortical neurons in the brain injury area of TBI rats, we performed TUNEL staining. As shown in Figure 8, the number of TUNEL-positive cells in the cerebral cortex of TBI +high dose rats was significantly reduced compared with that in the TBI +vehicle group.
Figure 8. Decreased neuronal apoptosis in TBI rats after application of CXCR2 antagonist SB225002. (A-E) The number of TUNEL-positive cells in the cortex of the injured area 3 days after injury in sham, TBI, TBI vehicle, TBI low dose, and TBI high dose. (F) The number of TUNEL-positive cells in the cortex of TBI rats in the injury area is significantly reduced after application of high dose of CXCR2 antagonist. Values are expressed as mean ± SEM. ***p < 0.001, vs. TBI vehicle; ###p < 0.001, vs. sham.
ERK, JNK, and NF-κB inhibitors downregulate mRNA expression of CXCL1, CXCR2 in TBI rats
To verify whether ERK, JNK, and NF-κB regulate the expression of CXCL1/CXCR2, the expression changes of CXCL1/CXCR2 were observed after application of their inhibitors PD98059, SP600125, and BAY117082, respectively. As shown in Figure 9, the mRNA expression of CXCL1 and CXCR2 decreased significantly after 3 days of continuous injection of high doses of ERK, JNK, and NF-κB inhibitors compared with the TBI +vehicle group, indicating that ERK, JNK, and NF-κB could regulate CXCL1 and CXCR2.
Figure 9. ERK, JNK, and NF-κB inhibitors PD98059, SP600125, and BAY117082 down-regulate CXCL1 and CXCR2 mRNA expression. (A) CXCL1 mRNA expression is decreased 3 days after continuous injection of high doses of ERK, JNK, and NF-κB inhibitors. (B) CXCR2 mRNA expression is decreased 3 days after continuous injection of high doses of ERK, JNK, and NF-κB inhibitors. Values are expressed as mean ± SEM. *** p < 0.001, **p < 0. 01, * p < 0.05, vs. TBI vehicle; ###p < 0.001, ##p < 0.01, vs. sham.
HBO improves neurological function in TBI rats
The effect of HBO therapy on the neurological function of TBI was observed by mNSS score. We found that after HBO treatment, the mNSS score of the TBI +HBO group was significantly lower than that of the TBI group at 3, 7, and 10 days after TBI, and HBO therapy could improve the neurological function of rats after TBI , shown in Figure 10.
Figure 10. HBO therapy improves neurological function in TBI rats. mNSS scores are lower in after TBI+HBO than TBI alone at 3, 7, and 10 days after TBI. Values are expressed as mean ± SEM. **p < 0.01, *p < 0. 05.
HBO treatment improved the cognitive function of rats after TBI
The Morris water maze was used to assess the effect of HBO on cognitive function in rats. In the locomotor navigation test, as shown in Figure 11A, the escape latency was significantly shorter in the TBI + HBO group than in the TBI group on days 5 and 6. In the 7-day spatial exploration trial after TBI, the number of platforms traversed by the TBI +HBO group was significantly higher than that of the TBI group. From these results, we can see that HBO treatment can improve the cognitive function of rats after TBI.
Figure 11. HBO treatment improves cognitive function in TBI rats. (A) In the locomotor navigation test, the escape latency is significantly lower after TBI+HBO than in TBI on days 5 and 6. (B) In the exploration test, the number of platform crossings is higher after TBI+HBO than TBI alone. Values are expressed as mean ± SEM. ***p < 0.001, *p < 0. 05.
HBO treatment inhibits neuronal apoptosis in rats with TBI
We evaluated the effect of HBO treatment on neuronal cell apoptosis in the cerebral cortex of rats with TBI. In Figure 12, the results show that the number of TUNEL-positive cells in the cerebral cortex of the TBI +HBO group was significantly reduced compared with the TBI group, indicating that HBO treatment could inhibit apoptosis of neuronal cells in TBI rats.
Figure 12. Decreased neuronal apoptosis in TBI rats after HBO treatment. (A-D) The number of TUNEL-positive cells (Bar=20 μm) in the cortex of the injured area 3 days after injury in sham, sham+HBO, TBI and TBI+HBO. (E) The number of TUNEL-positive cells in the cortex of TBI rats in the injury area after HBO treatment is significantly reduced compared to TBI alone. Values are expressed as mean ± SEM. *p < 0. 05.
HBO treatment downregulates CXCL1 and CXCR2 mRNA and protein expression after TBI in rats
To verify whether HBO treatment regulates expression of CXCL1/CXCR2, the mRNA and protein contents of CXCL1 and CXCR2 were detected by RT-qPCR and ELISA, respectively, after 3 days of continuous HBO therapy. In Figure 13, the results show that the mRNA and protein expression of CXCL1 and CXCR2 were significantly decreased compared with the TBI group, indicating that HBO treatment could down-regulate the expression of CXCL1 and CXCR2.
Figure 13. HBO treatment down-regulates mRNA and protein expression of CXCL1, CXCR2. (A) mRNA expression of CXCL1 and CXCR2 decreases after 3 days of HBO treatment. (B) protein expression of CXCL1 and CXCR2 decreases after 3 days of HBO treatment. Values are expressed as mean ± SEM. ***p < 0.001, *p < 0.05.
HBO treatment down-regulates the expression of p-ERK, p-JNK, and p-NF-κB after TBI in rats
To verify whether HBO therapy regulates CXCL1/CXCR2 expression by modifying ERK, JNK, and NF-κB, we tested the expression level of p-ERK, p-JNK, and p-NF-κB proteins by western blot after 3 days of continuous HBO treatment. In Figure 14, the results show that the levels of p-ERK, p-JNK, and p-NF-κB were significantly lower in the TBI +HBO group compared with the TBI group, suggesting that HBO treatment inhibits expression of CXCL1 and CXCR2 by downregulating the expression of p-ERK, p-JNK, and p-NF-κB.
Figure 14. HBO treatment down-regulates expression of ERK, JNK, and NF-κB. p-ERK, p-JNK, and p-NF-κB expression is significantly decreased after continuous HBO treatment for 3 days compared to TBI alone. Values are expressed as mean ± SEM. **p < 0.01; *p < 0.05.