Clemastine enhances myelin formation in the striatum and medial prefrontal cortex and improves sociability in a neonatal rat hypoxic-ischemic model

Fig. 1.. Schematic illustration (A) Schematic depiction of the development of the oligodendrocyte lineage. Oligodendrocyte precursor cells (OPCs) undergo proliferation and followed by differentiation into post-mitotic immature pre-oligodendrocytes. Under the specific condition, these immature oligodendrocytes can further mature into myelinating oligodendrocytes, which ensheath receptive axons and form compact myelin. (B) Schematic illustration of experimental timeline. Sham or neonatal hypoxia-ischemia (HI) was induced on postnatal day 10 in rats. Clemastine or vehicle was administered via oral gavage daily from postnatal day 11 to postnatal day 24. Rats were sacrificed on postnatal day 24 and postnatal day 35. Brain tissues were collected on postnatal day24. Three-chamber tests were conducted on postnatal day 35.

Fig. 2.. Clemastine reduced sociality deficits of neonatal hypoxia-ischemia (HI) rats on the postnatal day 35. (A) The paradigm for testing social preference and social novelty. (B) The average speed (m/s) among different groups in the three-chamber test. (C) Representative heat-map traces of rats exploring the two sides (Stranger 1 and empty cage). (D) Time spent in the compartment with the rat versus the empty compartment was measured. *p < 0.05. (E) The analysis of the social preference index in rats. (F) Representative traces of rats exploring the two sides (Stranger 1 and Stranger 2). (G) Time spent in the compartment with the novel stranger rat (stranger 2) or a familiar rat (stranger 1) was measured. Statistical analysis was performed using an unpaired t-test. *p < 0.05. (H) The social novelty index was analyzed using a one-way analysis of variance followed by post-hoc Bonferroni’s test. Data were expressed as the mean±SD. *p < 0.05 vs Sham group. #p < 0.05 vs HI group. n.s. indicates no significance.

Fig. 3.. Clemastin eattenuated myelin damage in the striatum and medial prefrontal cortex (mPFC) of HI rats on postnatal day 24. (A,B) Western blotting (left) and quantitative analyses (right) of the Myelin Basic Protein (MBP) protein expression in the striatum and mPFC on postnatal day 24. The protein expression was calculated by ImageJ analysis software and expressed as percentage changes versus the respective Sham group (n = 3–4). (C) Representative images showing MBP and NF200 immunoreactivity in the Striatum and mPFC. (D,E) The MBP/NF ratio in the striatum and mPFC was analyzed and expressed as percentage changes versus the respective Sham group (n = 6). (F) The correlation analysis between social novelty index and MBP protein expression in the striatum (spearman correlation, p = 0.0172,r = 0.7833). (G) The correlation analysis between social novelty index and MBP protein expression in the mPFC (spearman correlation, p = 0.0016, r = 0.8788). Data were expressed as the mean±SD and performed using a one-way analysis of variance followed by post-hoc Bonferroni’s test. *p < 0.05 vs Sham group. #p < 0.05 vs HI+Vehicle group.

Fig. 4.. Clemastine increased the number of oligodendrocytes in the striatum and mPFC of HI rats on postnatal day 24. (A) Representative immunofluorescence staining for CC1 (red) in the Striatum and mPFC, and nuclei were stained using DAPI. (B, C) The average number of CC1 positive cells per mm2 in the striatum and mPFC was calculated by ImageJ analysis software. Scale bar = 20 μm. All data were presented as mean ± SD (n = 6). * p < 0.05 versus Sham group; # p < 0.05 versus HI+Vehicle group.

Fig. 5.. Clemastine treatment suppressed HI-induced OPCs proliferation and apoptosis in the striatum of HI rats on postnatal day 24. (A) Representative immunofluorescence staining for Ki67 or cle-Caspase 3 (red) and PDGFRα (green) in the Striatum, and nuclei were stained using DAPI. (B) The average number of Ki67+ labeled OPCs (Ki67+ PDGFRα+ cells) was calculated by ImageJ analysis software (n = 6). (C) The average number of cle-Caspase 3+ labeled OPCs (cle-Caspase 3+ PDGFRα+ cells) was calculated by ImageJ analysis software (n = 6). (D) Western blotting (left) and quantitative analyses (right) of the PDGFRα protein expression in the striatum. The protein expression was calculated by ImageJ analysis software and expressed as percentage changes versus the respective Sham group (n = 4). Scale bar = 20 μm. All data were presented as mean ± SD (n = 6). * p < 0.05 versus Sham group; # p < 0.05 versus HI+Vehicle group.

Fig. 6.. Clemastine had no significant effect on OPCs in the mPFC area of HI rats on postnatal day 24. (A) Representative immunofluorescence staining for Ki67 or cle-Caspase (red) and PDGFRα (green) in the mPFC, and nuclei were stained using DAPI. (B) The average number of Ki67+ labeled OPCs (Ki67+ PDGFRα+ cells) was calculated by ImageJ analysis software (n = 6). (C)The average number of cle-Caspase 3+ labeled OPCs (cle-Caspase 3+ PDGFRα+ cells) was calculated by ImageJ analysis software (n = 6). (D) Western blotting (left) and quantitative analyses (right) of the PDGFRα protein expression in the mPFC. The protein expression was calculated by ImageJ analysis software and expressed as percentage changes versus the respective Sham group (n = 4). Scale bar = 20 μm. All data were presented as mean ± SD (n = 6). * p < 0.05 versus Sham group; # p < 0.05 versus HI+Vehicle group.

Fig. 7.. Clemastine treatment decreased the level of damaged myelin basic protein (dMBP) and microglia activation in the Striatum, but not mPFC.(A) Representative immunofluorescence staining for Iba1 (green, a marker of microglia), dMBP (red) in the striatum on postnatal day 24, and nuclei were stained using DAPI. (B,C) The dMBP area in the striatum and mPFC were calculated and expressed as percentage changes relative to the Sham group. (D,E) The microglial-associated dMBP in the striatum and mPFC were calculated and expressed as percentage changes relative to the Shamg roup. Scalebar= 20 μm. All data were presented as mean±SD(n = 6). *p < 0.05 versus Sham group; #p < 0.05 versus HI+Vehicle group.

Fig. 8.. Clemastine treatment reversed the activation of microglia and morphology change induced by HI in the striatum and mPFC. (A) Representative images of Iba1+ microglia. (B,C) The number of Iba1+ cells in the striatum and mPFC were analyzed and expressed as percentage changes relative to the Sham group. (D,E) The Iba1 staining area in the striatum and mPFC were calculated and expressed as percentage changes relative to the Sham group. (F) The sholl analysis of Iba1+ microglia. (G,H) The intersection number per radius over the distance from the cell body in the striatum and mPFC was displayed graphically in the curve. Scalebar= 20μm. All data were presented as mean±SD (n = 4–6). *p < 0.05 versus Sham group; #p < 0.05 versus HI+Vehicle group.

Fig. 9.. Diagram of the action of clemastine and neonatal hypoxia ischemic on myelin formation and sociability. Neonatal hypoxia-ischemia leads to hypomyelination in the striatum and mPFC, which in turn causes sociability deficits. Conversely, clemastine treatment enhances myelin formation and results in sociability improvement.