Objective: Iaccarino et al. (2016) [1] exposed 1 h of light flickering at 40 Hz to awake 5XFAD Alzheimer's Disease (AD) mouse models, generating action potentials at 40 Hz, activating~54% of microglia to colocalize with Ab plaque, acutely, and clearing~50% of Ab plaque after seven days, but only in the visual cortex. Hypothesis: Transcranially delivered, focused ultrasound (tFUS) can replicate the results of Iaccarino et al. (2016) [1] but throughout its area of application. Methods: We exposed sedated 5XFAD mice to tFUS (2.0 MHz carrier frequency, 40 Hz pulse repetition frequency, 400 ms-long pulses, spatial peak pulse average value of 190 W/cm 2 ). Acute studies targeted tFUS into one hemisphere of brain centered on its hippocampus for 1 h. Chronic studies targeted comparable brain in each hemisphere for 1 h/day for five days. Results: Acute application of tFUS activated more microglia that colocalized with Ab plaque relative to sham ultrasound (36.0 ± 4.6% versus 14.2 ± 2.6% [mean ± standard error], z ¼ 2.45, p < 0.014) and relative to the contralateral hemisphere of treated brain (36.0 ± 4.6% versus 14.3 ± 4.0%, z ¼ 2.61, p < 0.009). Chronic application over five days reduced their Ab plaque burden by nearly half relative to paired sham animals (47.4 ± 5.8%, z ¼ -2.79, p < 0.005). Conclusion:Our results compare to those of Iaccarino et al. (2016) [1] but throughout the area of ultrasound-exposed brain. Our results also compare to those achieved by medications that target Ab, but over a substantially shorter period of time. The proximity of our ultrasound protocol to those shown safe for non-human primates and humans may motivate its rapid translation to human studies.

Background and Purpose Immune responses to brain antigens after stroke contribute to poor outcome. We hypothesized that splenectomy would lessen the development of such responses and improve outcome. Methods Male Lewis rats (275–350 g) underwent 2 hrs middle cerebral artery occlusion (MCAO) immediately after splenectomy or sham-splenectomy. Animals were survived to 4 wks and immune responses to myelin basic protein (MBP) determined at sacrifice. Infarct volume was determined in a subset of animals sacrificed at 72 hrs. Behavioral outcomes were assessed to 672 hrs. Results Splenectomy was associated with worse neurological scores early after stroke, but infarct size at 72 hrs was similar in both groups. Behavioral outcomes and immune responses to MBP were also similar among splenectomized and sham-operated animals 672 hrs after MCAO. Conclusions Splenectomy did not alter the immune responses to brain antigens or improve outcome after stroke. Differences between this study and other studies of splenectomy and stroke are examined.

BackgroundMultiple sclerosis (MS) impacts approximately 400,000 in the United States and is the leading cause of disability among young to middle aged people in the developed world. Characteristic of this disease, myelin within generally focal volumes of brain tissue wastes away under an autoimmune assault, either inexorably or through a cycle of demyelination and remyelination. This centrally located damage produces central and peripheral symptoms tied to the portion of brain within the MS lesion site. Interestingly, Gibson and colleagues noted that optical activation of transgenically tagged central neurons increased the thickness of the myelin sheath around those neurons. Since ultrasound, delivered transcranially, can also activate brain focally, we hypothesized that ultrasound stimulation that followed the temporal pattern of Gibson et al. applied to MS lesions in a mouse model might either decelerate the demyelination phase or accelerate its remyelination phase.MethodsWe created a temporal pattern of ultrasound delivery that conformed to that of Gibson et al. and capable of activating mouse brain. We then applied ultrasound, transcranially, following that temporal pattern to separate cohorts of a mouse model of multiple sclerosis, using three different ultrasound carrier frequencies (0.625 MHz, 1.09 MHz, 2.0 MHz), during each of the demyelinating and remyelinating phases. After identifying the most promising protocol and MS brain state through qualitative analysis of myelin content, we performed additional studies for that condition then assayed for change in myelin content via quantitative analysis.ResultsWe identified one ultrasound protocol that significantly accelerated remyelination, without damage, as demonstrated with histological analysis.ConclusionMRI-guided focused ultrasound systems exist that can, in principle, deliver the ultrasound protocol we successfully tested here. In addition, MRI, as the clinical gold standard, can readily identify MS lesions. Given the relatively low intensity values of our ultrasound protocol – close to FDA limits – we anticipate that future success with this approach to MS therapy as tested using more realistic MS mouse models may one day translate to clinical trials that help address this devastating disease.

Summary Zebrafish regenerate fin rays following amputation through epimorphic regeneration, a process that has been proposed to involve the epithelial-to-mesenchymal transition (EMT). We performed single-cell RNA sequencing (scRNA-seq) to elucidate osteoblastic transcriptional programs during zebrafish caudal fin regeneration. We show that osteoprogenitors are enriched with components associated with EMT and its reverse, mesenchymal-to-epithelial transition (MET), and provide evidence that the EMT markers cdh11 and twist2 are co-expressed in dedifferentiating cells at the amputation stump at 1 dpa, and in differentiating osteoblastic cells in the regenerate, the latter of which are enriched in EMT signatures. We also show that esrp1 , a regulator of alternative splicing in epithelial cells that is associated with MET, is expressed in a subset of osteoprogenitors during outgrowth. This study provides a single cell resource for the study of osteoblastic cells during zebrafish fin regeneration, and supports the contribution of MET- and EMT-associated components to this process.

While humans have limited potential for limb regeneration, some vertebrates can regenerate bony appendages following amputation. During zebrafish fin regeneration, mature osteoblasts at the amputation stump dedifferentiate and migrate to the blastema, where they re-enter the cell cycle and then re-differentiate to form new bone. Osteoblastic cells exhibit dual mesenchymal and epithelial characteristics during fin regeneration, however little is known about why or how this occurs. Using single-cell RNA-sequencing, we found osteoprogenitors are enriched with components associated with the epithelial-to-mesenchymal transition (EMT) and its reverse, mesenchymal-to-epithelial transition (MET). In trajectory analyses, osteoblastic cells solely expressed EMT components, or transiently expressed MET components prior to expressing those for EMT. We found that cdh11, a cancer EMT marker, is expressed during osteoblast dedifferentiation. We also found that esrp1, a regulator of alternative splicing in epithelial cells whose expression is important for MET, is expressed in a subset of osteoprogenitors during outgrowth. This study provides a valuable single cell resource for the study of osteoblast differentiation during zebrafish fin regeneration, and identifies MET- and EMT-associated components which may be important for successful appendage regeneration.

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