Intranasal mesenchymal stem cell treatment for neonatal brain damage: long-term cognitive and sensorimotor improvement

Abstract

Mesenchymal stem cell (MSC) administration via the intranasal route could become an effective therapy to treat neonatal hypoxic-ischemic (HI) brain damage. We analyzed long-term effects of intranasal MSC treatment on lesion size, sensorimotor and cognitive behavior, and determined the therapeutic window and dose response relationships. Furthermore, the appearance of MSCs at the lesion site in relation to the therapeutic window was examined. Nine-day-old mice were subjected to unilateral carotid artery occlusion and hypoxia. MSCs were administered intranasally at 3, 10 or 17 days after hypoxia-ischemia (HI). Motor, cognitive and histological outcome was investigated. PKH-26 labeled cells were used to localize MSCs in the brain. We identified 0.5 × 10(6) MSCs as the minimal effective dose with a therapeutic window of at least 10 days but less than 17 days post-HI. A single dose was sufficient for a marked beneficial effect. MSCs reach the lesion site within 24 h when given 3 or 10 days after injury. However, no MSCs were detected in the lesion when administered 17 days following HI. We also show for the first time that intranasal MSC treatment after HI improves cognitive function. Improvement of sensorimotor function and histological outcome was maintained until at least 9 weeks post-HI. The capacity of MSCs to reach the lesion site within 24 h after intranasal administration at 10 days but not at 17 days post-HI indicates a therapeutic window of at least 10 days. Our data strongly indicate that intranasal MSC treatment may become a promising non-invasive therapeutic tool to effectively reduce neonatal encephalopathy.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Dose effect of MSCs on motor performance and lesion volume at 35 days post-HI.

Mice received 0.25×106, 0.5×106, 1×106 MSCs or Vehicle (Veh) treatment at 10 days after induction of HI. (A) Paw preference to use the unimpaired forepaw in the cylinder rearing test (CRT) was assessed at 5 weeks post-HI. Sham-operated littermates (Sham) were used as controls. Quantification of ipsilateral MAP2 (B) and MBP(C) area loss measured as 1- (ipsi-/contralateral MAP2- or MBP-positive area) at 5 weeks post-HI. (D) Representative sections of MAP2 loss. Insets show higher magnification of corresponding MAP2 sections. Scale bar = 100 µm. (E) Representative sections of MBP area loss. Data represent mean ± SEM. Sham n = 8; Veh n = 10; 0.25×106 MSC n = 11; 0.5×106 MSC n = 10; 1×106 MSC n = 13. *p<0.05; **p<0.01 vs Veh. Data presented in this figure are results from pooled experiments out of 8 different litters. Treatment groups were randomly distributed between litters.

Figure 2. Therapeutic window for MSC treatment.

Mice received 0.5×106 MSCs or Veh at 3, 10 or 17 days post-HI. Because no significant difference was found between Veh groups treated at different time points we pooled all animals into one group. (A) Unimpaired forepaw preference in the CRT at 5 weeks post-insult. Quantification of ipsilateral MAP2 (B) and MBP (C) area loss measured as 1- (ipsi-/contralateral MAP2- or MBP-positive area) at 5 weeks post-insult. Insets show representative examples of MAP2 or MBP staining. Data represent mean ± SEM. Sham n = 8; Veh n = 19; MSC 3 d n = 12; MSC 10 d n = 17; MSC 17 d n = 9; *p<0.05; **p<0.01; ***p<0.001 vs. Veh. Data presented in this figure are results from pooled experiments out of 12 different litters. Treatment groups were randomly distributed between litters.

Figure 3. Effect of two MSC treatments on sensorimotor function and lesion size.

Mice received 0.5×106 MSCs or Veh at 3, 10 or 3+10 days post-insult. Because no significant difference was found between Veh groups treated at different time points we pooled all animals into one group. (A) Unimpaired forepaw preference in the CRT at 5 weeks post-HI. Quantification of ipsilateral MAP2 (B) and MBP (C) area loss measured as 1- (ipsi-/contralateral MAP2- or MBP-positive area) at 5 weeks post-HI. Insets show representative examples of MAP2 or MBP staining. Data represent mean ± SEM. Sham n = 7; Veh n = 19; MSC 3+10 d n = 13; MSC 10+17 d n = 11; MSC 10 d n = 17. ***p<0.001 vs. Veh., n.s. = non-significant. Data presented in this figure are results from pooled experiments out of 14 different litters. Treatment groups were randomly distributed between litters.

Figure 4. Long-term effect of MSC treatment on sensorimotor function and lesion volume.

Mice received 0.5×106 MSCs or Veh at 3, 10 or 3+10 days post-HI. Because no significant difference was found between Veh groups treated at different time points we pooled all animals into one group. (A) Unimpaired forepaw preference in the CRT at 8 weeks post-HI. Quantification of ipsilateral MAP2 (B) and MBP (C) area loss measured as 1- (ipsi-/contralateral MAP2- or MBP-positive area). Insets show representative examples of MAP2 or MBP staining at 9 weeks post-insult. Data represent mean ± SEM. Sham n = 23; Veh n = 23; MSC 10 d n = 23; MSC 3+10 d n = 12. *p<0.05; **p<0.01 vs. Veh. Data presented in this figure are results from pooled experiments out of 13 different litters. Treatment groups were randomly distributed between litters.

Figure 5. Effect of MSC treatment on cognitive behavior.

Mice received 0.5×106 MSCs or Veh at 10 or 3+10 days post-HI. Because no significant difference was found between Veh groups treated at different time points we pooled all animals into one group. Animals were tested for cognitive function using the social discrimination test at 7 weeks post-HI. Preference for novel conspecific is expressed as exploratory ratio. The total social interaction time did not differ between groups. (A) Preference for novel mouse after a 5 min interval. (B) Preference for novel mouse after a 3 hour interval. (C)Training session to measure preference for social novelty as an indication for social avoidance. Sham n = 23; Veh n = 23; MSC 10 d n = 23; MSC 3+10 d n = 12. *p<0.05; **p<0.01 in relation to 50% (no discrimination); #p<0.05; ##p<0.01 vs. Veh. Data presented in this figure are results from pooled experiments out of 13 different litters. Treatment groups were randomly distributed between litters.

Figure 6. Presence of MSCs in the brain.

PKH-26 labeled 1.0×106 MSCs were administered intranasally at 3, 10 and 17 days post-HI. Because no significant difference was found between Veh groups treated at different time points we pooled all animals into one group. (A, B, C) Notice the severe HI-induced damage, as the layer structure of the ipsilateral cortex and hippocampus are lost. (A) MSCs (red) in the ipsilateral hippocampus (see arrow heads) 24 h after administration at 3 days post-HI. (B) MSCs (see arrow heads) in the ipsilateral damaged cortex 24 h after administration at 10 days post-HI. (C) Lack of MSCs (see arrow heads) in the ipsilateral cortical areas surrounding the lesion site when MSCs are given at 17 days post-HI. Contralateral pictures depict hippocampal area (in A) and cortical area (in B and C). (D) Control groups showing lack of MSCs in the hippocampal area and cortical area at 3 and 10 days, respectively, after MSC administration in sham-operated animals and HI-Vehicle treated brain without MSC treatment. Asterisk = lesion site. Blue = Dapi staining. Scale bar 50 µm. Data presented in this figure are results from pooled experiments out of 10 different litters. Treatment groups were randomly distributed between litters.