Figure 1.
(A) For the hEPO+MBs/FUS group in the acute phase study, hEPO (i.v. injection) and first MBs/FUS were applied approximately 5 h after a 50-min 3VO. A second MBs/FUS was given 15 min later. Animals were sacrificed 24 h after 3VO. (B) In the chronic study, experimental groups were tested for motor deficits with gait analysis and cylinder test at indicated time points. Animals were sacrificed on Day 28. (C) Coronal brain section showed the cortex locations with a 2-mm interval for the two MBs/FUS application.
Figure 2.
Enhancement of hEPO delivery into the brain tissues by MBs/FUS.
hEPO and MBs were intravenously administered and FUS was transcranially applied. (A) Rat brains were perfused and sliced into sections at 3 h after hEPO injection. Sonicated region of the brain was dissected and quantified. The hEPO levels in sections 3 and 4 were significantly higher in the I/R+hEPO+MBs/FUS group compared to the I/R+hEPO group (n = 3 for each). Data were given as means ± S.E.M., * p<0.05, as compared with the I/R+hEPO group. (B) CSF was sampled at 3 h after hEPO injection. The hEPO concentration of CSF in the I/R+hEPO+MBs/FUS group showed significant enhancement compared with the I/R+hEPO group. (C) The serum hEPO was also sampled at 3 h after hEPO injection. No hEPO was found in the sham and I/R groups without hEPO injection.
Figure 3.
Reduction of brain infarct volume and neurological score by hEPO+MBs/FUS in rat experiments.
(A) All rats were induced ischemia by 3VO for 50 min. The brain was removed and stained with TTC 24 h after 3VO. Representative sections of TTC-stained coronal brain showed the infarct area 24 h after 3VO for the I/R (control), I/R+MBs/FUS, I/R+hEPO, and I/R+hEPO+MBs/FUS groups. (B) Infarct volume was presented as the percentage of contralateral cortex. Note that the infarct volume was significantly reduced in the hEPO+MBs/FUS group. (C) Neurological score was performed 24 h after I/R. Note that hEPO combined with MBs/FUS significantly reduced the neurological scores. Data were presented as mean ± SEM, * p<0.05 as compared with the control group.
Figure 4.
hEPO+MBs/FUS inhibits the ischemia/reperfusion-induced neuronal death and inflammation in rat experiments.
Immunohistochemical staining of NeuN, CD-11b, and GFAP was performed 24 h after I/R. (A) illustrated the position of FUS sonication. In NeuN staining (B, E, and H), the I/R group showed a marked loss of neurons, whereas, neurons were intact in the sham and I/R+hEPO+MBs/FUS groups. In CD-11b staining (C, F, and I), the I/R group showed microglia activation (condensed nuclei), whereas the sham and I/R+hEPO+MBs/FUS groups showed ramified microglia. In GFAP staining (D, G, and J), there was an increase of GFAP in the I/R group but not in the sham and I/R+hEPO+MBs/FUS groups. (Scale bar = 200 µm).
Figure 5.
Increase of residual brain volume and improvement of limb-use in chronic phase of 3VO by hEPO+MBs/FUS.
(A) Representative Nissl staining showed the residual neurons 28 days after I/R. (Scale bar = 5 mm) (B) Residual brain volume was presented as the percentage of contralateral cortex. 28 days after I/R, residual brain volume was reduced markedly, while it was antagonized by I/R+hEPO+MBs/FUS. (C) The cylinder test was used to evaluate the rat behavior after brain injury. The I/R group showed a deficit in the usage of the left forepaw from Day-3 to Day-28, while it was reversed by hEPO+MBs/FUS. The average percentage of paw usage in normal rats was 50%. Data were presented as mean ± SEM (n = 5 for each group), * p<0.05 as compared with sham control group, # p<0.05 as compared with the I/R group.
Figure 6.
Improvement in catwalk automated gait analysis test by hEPO plus MBs/FUS in chronic phase of 3VO.
Two gait analysis parameters, paw intensity (A) and paw angle (B), were assessed from Day-7 to Day-28 after I/R. In both paw intensity and paw angle, treatment with hEPO+MBs/FUS significantly improved the performance of impaired limb. Data were shown as mean ± SEM (n = 5 for each group), * p<0.05 as compared with the sham (control group), # p<0.05 as compared with the I/R group.