Focused Ultrasound-Induced Neurogenesis Requires an Increase in Blood-Brain Barrier Permeability

<div><p>Transcranial focused ultrasound technology used to transiently open the blood-brain barrier, is capable of stimulating hippocampal neurogenesis; however, it is not yet known what aspects of the treatment are necessary for enhanced neurogenesis to occur. The present study set out to determine whether the opening of blood-brain barrier, the specific pressure amplitudes of focused ultrasound, and/or the intravenous administration of microbubbles (phospholipid microspheres) are necessary for the enhancement of neurogenesis. Specifically, mice were exposed to burst (10ms, 1Hz burst repetition frequency) focused ultrasound at the frequency of 1.68MHz and with 0.39, 0.78, 1.56 and 3.0MPa pressure amplitudes. These treatments were also conducted with or without microbubbles, at 0.39 + 0.78MPa or 1.56 + 3.0MPa, respectively. Only focused ultrasound at the ~0.78 MPa pressure amplitude with microbubbles promoted hippocampal neurogenesis and was associated with an increase in blood-brain barrier permeability. These results suggest that focused ultrasound -mediated neurogenesis is dependent upon the opening of the blood-brain barrier.</p></div

Effects of Neurotrophic Support and Amyloid-Targeted Combined Therapy on Adult Hippocampal Neurogenesis in a Transgenic Model of Alzheimer's Disease

<div><p>Although it is recognized that multi-drug therapies may be necessary to combat AD, there is a paucity of preclinical proof of concept studies. We present a combination treatment paradigm, which temporally affects different aspects of Alzheimer’s disease (AD)-like pathology, specifically Aβ-toxicity and neurogenesis. At early stages of AD-like pathology, in TgCRND8 mice, we found that combating Aβ pathology with <i>scyllo</i>-inositol ameliorated deficits in neurogenesis. Older TgCRND8 mice with established amyloid load had decreased progenitor cell proliferation and survival compared to non-transgenic mice, regardless of <i>scyllo</i>-inositol treatment. The prolonged exposure to Aβ-pathology leads to deficits in the neurogenic niche, thus targeting Aβ alone is insufficient to rescue neurogenesis. To support the neurogenic niche, we combined <i>scyllo</i>-inositol treatment with leteprinim potassium (neotrofin), the latter of which stimulates neurotrophin expression. We show that the combination treatment of <i>scyllo</i>-inositol and neotrofin enhances neuronal survival and differentiation. We propose this proof of concept combination therapy of targeting Aβ-pathology and neurotrophin deficits as a potential treatment for AD.</p></div

Timeline of therapeutic interventions.

<p>(A) TgCRND8 (Tg) mice with early AD-like pathology and age-matched non-transgenic (NTg) mice were treated or untreated from 72 days of age until sacrifice at either 100 or 121 days, for proliferation and differentiation/survival, respectively. BrdU was intraperitoneally injected in these cohorts of mice for 5 days from day 96–100. (B) Tg mice with late AD-like pathology and age-matched NTg mice were treated or untreated from day 172 days of age until sacrifice at either 200 or 221 days. Tg mice were treated with <i>scyllo</i>-inositol (Tg-SI), neotrofin (Tg-NEO) or a combination of both (Tg-SI/NEO) for 28 days. BrdU was injected in these cohorts of mice for 5 days from day 196–200.</p

The average number of mature and immature neurons colabelled with BrdU in each slice of the dentate gyrus.

<p>Animals were from group 1 (~0.78 MPa pressure amplitudes with microbubbles). Error bars represent the standard error of the mean. † = 0.061, *<i>p</i> = 0.021.</p

Hippocampal cell proliferation, differentiation and survival in Tg mice with early AD-like pathology and age-matched Tg littermates.

<p>(A) A representative image of the dentate gyrus stained for BrdU, demonstrates the distribution of proliferating BrdU+ cells. (B) A representative image of BrdU (green), DCX (red) and GFAP (white) positive cells in the dentate gyrus at 100 days of age. The arrow highlights a representative BrdU+/DCX+ cell. (C) A representative image of BrdU (green), NeuN (red) and GFAP (white) positive cells in the dentate gyrus. The arrow highlights a representative BrdU+/NeuN+ cell. Cell proliferation was examined at 100 days of age (D,E) while differentiation and survival was examined at 121 days of age (F,G) as a function of <i>scyllo</i>-inositol treatment. (D) The number of proliferating BrdU+ cells in the dentate gyrus of NTg (n = 7), NTg-SI (n = 6), Tg (n = 7) and Tg-SI (n = 5) mice were compared. This demonstrates more BrdU+ cells in Tg compared to NTg and to Tg-SI. (E) The percentage of BrdU+/DCX+ cells in the dentate gyrus showed less neuronal BrdU+ cells in Tg compared to the other three cohorts. (F) The number of BrdU+ cells surviving in NTg (n = 6), NTg-SI (n = 5), Tg (n = 6) and Tg-SI (n = 6) mice demonstrates more cell survival in Tg-SI mice. (G) The percentage of BrdU+/NeuN+ cells shows no difference between cohorts. Scale bars indicate 100 μm (A) or 25 μm (B,C). Data are mean ± SEM. One-way ANOVA with Fisher’s Post-hoc test, * represents p< 0.05 and ** represents p< 0.01.</p

Focused Ultrasound-Induced Neurogenesis Requires an Increase in Blood-Brain Barrier Permeability - Fig 1

<p>Representative post-sonication T1-weighted MR images of animals in (A) group 1 (~0.78 MPa pressure amplitudes with microbubbles), (B) group 2 (0.39MPa pressure amplitudes with microbubbles), (C) group 3 (1.56MPa pressure amplitudes without microbubbles), and (D) group 4 (3.0MPa pressure amplitudes without microbubbles). Only animals that received ~0.78MPa pressure amplitudes and microbubbles showed significant opening of the blood-brain barrier. Arrow indicates area of BBB opening.</p

Hippocampal cell differentiation and survival in <i>scyllo</i>-inositol, neotrofin, and <i>scyllo</i>-inositol/neotrofin treated 200 day old Tg mice.

<p>(A) The number of DCX+ cells in Tg-SI (n = 6), Tg-NEO (n = 6) and Tg-SI/NEO (n = 7) mice were not significantly different between cohorts. (B) The percentage of DCX+ cells that were DCX+/CR+ immature neurons were assessed and showed no difference between treatment groups. (C) The percentage of DCX+/CR+ cells that were NeuN+ represents immature neurons approaching maturity. Tg-SI/NEO mice had a significantly greater percentage of DCX+/CR+/NeuN+ cells than Tg-SI and Tg-NEO mice. Tg-SI mice had a greater percentage than Tg-NEO mice. (D/E) Representative images of DCX (green) and CR (red) positive cells in the hippocampus, showing DCX+/CR- and DCX+/CR+ cells in Tg-SI mice and in Tg-SI/NEO mice. (E) Arrows indicate DCX+/CR+ cells. Scale bar indicates 20 μm. Data are mean ± SEM. One-way ANOVA with Fisher’s Post-hoc test, *** represents p<0.001.</p

Hippocampal cell proliferation in Tg mice with late AD-like pathology and age-matched NTg littermates.

<p>Cell proliferation was assessed at 200 days of age (A,B). (A) The number of proliferating BrdU+ cells in NTg (n = 6), NTg-SI (n = 6), Tg (n = 6) and Tg-SI (n = 5) mice was compared, demonstrating less proliferation in Tg and Tg-SI vs. NTg-SI. (B) The percentage of BrdU+/DCX+ cells in the dentate gyrus was not different between cohorts. (C) Dentate gyrus stained with BrdU (red) and DCX (green) demonstrating the distribution of proliferating cells, as well as neuroblasts and immature neurons, respectively. (D) Representative orthogonal projection of a BrdU+/DCX+ cell. Scale bar indicates 100 μm (C) or 25 μm (D). Data are mean ± SEM. One-way ANOVA with Fisher’s Post-hoc test, ** represents p< 0.01.</p

Phenotypic characterization of total CR cells in the subgranular zone and granular cell layer of the dentate gyrus in Tg mice treated with <i>scyllo</i>-inositol, neotrofin or <i>scyllo</i>-inositol/neotrofin.

<p>Phenotypic characterization of total CR cells in the subgranular zone and granular cell layer of the dentate gyrus in Tg mice treated with <i>scyllo</i>-inositol, neotrofin or <i>scyllo</i>-inositol/neotrofin.</p

Specifications of focused ultrasound treatments for each group.

<p>Specifications of focused ultrasound treatments for each group.</p