Ultrasound-microbubble enhances bioavailability of neuro growth factor in neuro-retina after intravitreal injection in rabbits

This study was to determine and compare the distribution and concentration of mNGF between two groups with and without ultrasound microbubble in rabbit's eyes after intravitreal injection. Intravitreal injection of mNGF (18 mu g/100 mu L) with and without mixing of microbubbles were performed on 48 New Zealand rabbits (96 eyes). The left eyes (48 eyes, group A) were intravitreally injected with 18 mu g/100 mu L of mNGF only at supertemporal sclera apart from 3 mm cornea sclera edge. The right eyes (48 eyes, group B) were intravitreally injected with SonoVue (100 mu L), a type of microbubble, followed by 100 mu L of mNGF at the same time. After the injections, the right eyes (group B) were immediately radiated with ultrasound of 1 MHZ frequency, 0.5 W/cm(2) ultrasonic intensity for 60 s. Then, the rabbits were sacrificed at points of 0.5, 1, 2, 3, 4, 6, 12 and 24 hours after injection, with 6 rabbits at each time point. The eye tissue was collected for determination of mNGF concentration in dissected ocular tissue of vitreous body, retina and optic nerve by high performance liquid chromatography (HPLC). After the injection of same amount of mNGF with or without SonoVue and ultrasound, the concentration of mNGF in vitreous decreased lineally with the time elapsed. The kinetics followed the pattern of first-order. In group A, the concentration of mNGF changed from 2.186 +/- 0.089 ng/mg to 0.061 +/- 0.001 ng/mg without SonoVue and ultrasound, and in group B, the concentration of mNGF changed from 1.949 +/- 0.048 ng/mg to 0.058 +/- 0.002 ng/mg with SonoVue and ultrasound. Concerning the concentration of mNGF, there is significant difference between two groups at 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 24 h after injection (P=0.000, 0.001, 0.000, 0.021, 0.047, 0.008 and 0.012, respectively). The injection of mNGF with SonoVue and ultrasound resulted in a quicker pervasion and a lower concentration in vitreous than the injection of mNGF only at all the time points. The distribution of mNGF in retina and optic nerve after the injection with or without SonoVue followed a two-phase pattern. Without SonoVue and ultrasound in group A, the mean values of concentration of mNGF were 0.152 +/- 0.010 ng/mg, 0.193 +/- 0.008 ng/mg, 0.257 +/- 0.011 ng/mg, 0.385 +/- 0.013 ng/mg, 0.277 +/- 0.014 ng/mg, 0.180 +/- 0.007 ng/mg, 0.064 +/- 0.010 ng/mg and 0.002 +/- 0.000 ng/mg at 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 12 h and 24 h, respectively in retina; and they were 0.080 +/- 0.003 ng/mg, 0.110 +/- 0.009 ng/mg, 0.148 +/- 0.007 ng/mg, 0.222 +/- 0.012 ng/mg, 0.246 +/- 0.010 ng/mg, 0.122 +/- 0.004 ng/mg, 0.029 +/- 0.008 ng/mg and 0.000 +/- 0.000 ng/mg at 0.5 h, 1 h, 2 h, 3h, 4 h, 6 h, 12 h and 24 h, respectively in optic nerve. With SonoVue and ultrasound in group B, all of the mean value (except at 24 h due to its value of 0.000 +/- 0.000 ng/mg in optic nerve) was higher than that in group A, which was 0.194 +/- 0.012 ng/mg (P=0.004), 0.228 +/- 0.007 ng/mg (P=0.000), 0.316 +/- 0.012 ng/mg (P=0.000), 0.442 +/- 0.011 ng/mg (P=0.002), 0.306 +/- 0.008 ng/mg (P=0.000), 0.193 +/- 0.005 ng/mg (P=0.000), 0.083 +/- 0.004 ng/mg (P=0.000) and 0.003 +/- 0.000 ng/mg (P=0.000) at 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 12 h and 24 h, respectively in retina; and they were 0.101 +/- 0.006 ng/mg (p=0.000), 0.141 +/- 0.006 ng/mg (P=0.000), 0.189 +/- 0.014 ng/mg (P=0.002), 0.257 +/- 0.004 ng/mg (P=0.001), 0.301 +/- 0.012 ng/mg (P=0.001), 0.140 +/- 0.005 ng/mg (P=0.001), 0. 042 +/- 0.007 ng/mg (P=0.001) and 0.000 +/- 0.000 ng/mg at 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 12 h and 24 h, respectively in optic nerve. The differences in all time point between group A and B in retina were statistical significance, while in all time point except for the time point of 24 h in optic nerve were also statistical significance (P < 0.05). The content reached a peak in 3 hour in retina (0.385 +/- 0.013 ng/mg in group A, 0.442 +/- 0.011 ng/mg in group B) and in 4 hour in optical nerve (0.246 +/- 0.010 ng/mg in group A, 0.301 +/- 0.012 ng/mg in group B) after the injection in both groups. At the time point of 12 hour, there was a 1.29 fold higher content in retina in the group with microbubble than that without the microbubble, and there was a 1.44 fold higher content in the optic nerve in the group with microbubble than that without the microbubble. The higher concentration of the mNGF at whole time course in both retina and optic nerve was always associated with the injection of the combined mNGF and SonoVue, mediated by ultrasound. The allocation of the mNGF maintained a longer and higher existing of the agent in retina than that in optic nerve. Intravitreal injection of mNGF together with ultrasound microbubble delivery can generated a higher concentration in the target tissue of retina and optic nerve than the injection with mNGF only. Ultrasound-microbubble enhances bioavailability of mNGF in neuro-retina after intravitreal injection in rabbits.