Fig. 1
Plasma_EVCCI damages neuronal dendritic spines, and the EV inhibitor GW4869 alleviates hyperalgesia and memory impairment in CCI rats. Plasma EVs were extracted by differential high-speed centrifugation of the rats’ plasma. (A) NTA analysis showed that the concentration and diameter of plasma EVs were greater in CCI rats than in sham rats. n = 9 rats. unpaired Student’s t test was used. (B, C) DIL staining and immunofluorescence staining showed that plasma_EVCCI  significantly inhibited the dendritic spine density and the expression levels of PSD95 and SYN proteins in primary hippocampal neurons. n = 4–6 images from three wells of each group. unpaired Student’s t test was used. (D) Flowchart for establishing the CCI model, GW4869 intervention, evaluation of nociceptive hypersensitivity and memory behavior evaluation; The paw withdrawal threshold (PWT), and paw withdrawal latency (PWL) were each determined using Von Frey filaments and thermal pain stimulators, respectively. GW4869 treatment substantially raised the PWT and PWL at 1, 3, 7, 14 and 21 days after CCI. n = 12 rats, two-way ANOVA followed by Tukey’s multiple comparisons test was used. (E) Compared with CCI or vehicle treatment, GW4869 treatment enhanced the spontaneous alternation behavior of CCI rats. n = 10 rats. two-way ANOVA followed by Tukey’s multiple comparisons test was used. (F) Compared with CCI or vehicle treatment, GW4869 treatment increased the recognition indices of CCI rats in the ORM, OLM and TOM tests. n = 8–10 rats. two-way ANOVA followed by Tukey’s multiple comparisons test was used. (G, H) After Golgi staining, Sholl analysis and ImageJ analysis showed that GW4869 rescued the dendritic complexity, dendritic length, density, and spine head size in hippocampal neurons from CCI rats compared with neurons from the hippocampus of CCI or CCI + Vehicle rats. n = 5–6 stained sections from 3 animals in each group. ordinary one-way followed by Tukey’s multiple comparisons test was used for dendritic length; mixed-effects analysis followed by Tukey’s multiple comparisons test was used for dendritic density and spine head size; two-way ANOVA followed by Tukey’s multiple comparisons test was used for dendritic complexity (I) GW4869 improved PSD95, SYN and BDNF protein levels in the hippocampus of CCI rats. The data are shown as the means ± SDs. ordinary one-way followed by Tukey’s multiple comparisons test was used. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001