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An emerging approach with potential in improving the treatment of neurodegenerative diseases and brain tumors is the use of focused ultrasound (FUS) to bypass the blood–brain barrier (BBB) in a non-invasive and localized manner. A large body of pre-clinical work has paved the way for the gradual clinical implementation of FUS-induced BBB opening. Even though the safety profile of FUS treatments in rodents has been extensively studied, the histological and behavioral effects of clinically relevant BBB opening in large animals are relatively understudied. Here, we examine the histological and behavioral safety profile following localized BBB opening in non-human primates (NHPs), using a neuronavigation-guided clinical system prototype. We show that FUS treatment triggers a short-lived immune response within the targeted region without exacerbating the touch accuracy or reaction time in visual-motor cognitive tasks. Our experiments were designed using a multiple-case-study approach, in order to maximize the acquired data and support translation of the FUS system into human studies. Four NHPs underwent a single session of FUS-mediated BBB opening in the prefrontal cortex. Two NHPs were treated bilaterally at different pressures, sacrificed on day 2 and 18 post-FUS, respectively, and their brains were histologically processed. In separate experiments, two NHPs that were earlier trained in a behavioral task were exposed to FUS unilaterally, and their performance was tracked for at least 3 weeks after BBB opening. An increased microglia density around blood vessels was detected on day 2, but was resolved by day 18. We also detected signs of enhanced immature neuron presence within areas that underwent BBB opening, compared to regions with an intact BBB, confirming previous rodent studies. Logistic regression analysis showed that the NHP cognitive performance did not deteriorate following BBB opening. These preliminary results demonstrate that neuronavigation-guided FUS with a single-element transducer is a non-invasive method capable of reversibly opening the BBB, without substantial histological or behavioral impact in an animal model closely resembling humans. Future work should confirm the observations of this multiple-case-study work across animals, species and tasks.

Focused ultrasound (FUS) has been used to non-invasively elicit or inhibit motor neuronal activity in the mouse peripheral nervous system in vivo. However, less is known about whether FUS elicits immune system responses associated with peripheral sensory neuronal activity. In this study, we sought to determine that non-invasive ultrasound image-guided FUS can elicit the neurogenic axon reflex of peripheral nerves in the mouse sciatic nerve. The local vasodilation in the plantar view of the hind paw detected with a high-resolution laser Doppler imager indicated neurogenic flare responses after FUS stimulation. The effects of FUS were compared with control groups, where a distinct pattern of blood flow changes was observed only in FUS-elicited neurogenic flare responses. The findings indicate that image-guided FUS elicits local axon reflexes in vivo with a high degree of specificity and penetration depth.

International audience; The blood-brain barrier is the primary obstacle to efficient intracerebral drug delivery. Focused ultrasound, in conjunction with microbubbles, is a targeted and non-invasive way to disrupt the blood-brain barrier. Many commercially available ultrasound contrast agents and agents specifically designed for therapeutic purposes have been investigated in ultrasound-mediated blood-brain barrier opening studies. The new generation of sono-sensitive agents, such as liquid-core droplets, can also potentially disrupt the blood-brain barrier after their ultrasound-induced vaporization. In this review, we describe the different compositions of agents used for ultrasound-mediated blood-brain barrier opening in recent studies, and we discuss the challenges of the past five years related to the optimal formulation of agents.

International audience; Since its discovery in 2001, the interest in the low-intensity focused ultrasound (FUS)-mediated blood-brain barrier (BBB) disruption to deliver genes and drugs to brain tissue has increased steadily. Increasingly sophisticated sonication protocols and dedicated hardware are being developed to efficiently and safely permeabilize the BBB, and novel magnetic resonance (MR)-based technologies have been designed to guide FUS-induced BBB opening protocols. MR imaging (MRI) allows not only to more precisely target brain regions and evaluate the outcome of sonication in terms of enhanced BBB permeability but also to control the effects of ultrasound on brain structure and function. This review summarizes the state of the art in current MRI hardware and methods used in BBB opening protocols both in pre-clinical and clinical settings.

Ultrasound can modulate activity in the central nervous system, including the induction of motor responses in rodents. Recent studies investigating ultrasound-induced motor movements have described mostly bilateral limb responses, but quantitative evaluations have failed to reveal lateralization or differences in response characteristics between separate limbs or how specific brain targets dictate distinct limb responses. This study uses high-resolution focused ultrasound (FUS) to elicit motor responses in anesthetized mice in vivo and four-limb electromyography (EMG) to evaluate the latency, duration and power of paired motor responses (n = 1768). The results indicate that FUS generates target-specific differences in electromyographic characteristics and that brain targets separated by as little as 1 mm can modulate the responses in individual limbs differentially. Exploiting these differences may provide a tool for quantifying the susceptibility of underlying neural volumes to FUS, understanding the functioning of the targeted neuroanatomy and aiding in mechanistic studies of this non-invasive neuromodulation technique.

Focused ultrasound (FUS)-mediated blood-brain barrier (BBB) opening is currently being investigated in clinical trials. Here, we describe a portable clinical system with a therapeutic transducer suitable for humans, which eliminates the need for in-line magnetic resonance imaging (MRI) guidance. A neuronavigation-guided 0.25-MHz single-element FUS transducer was developed for non-invasive clinical BBB opening. Numerical simulations and experiments were performed to determine the characteristics of the FUS beam within a human skull. We also validated the feasibility of BBB opening obtained with this system in two non-human primates using U.S. Food and Drug Administration (FDA)-approved treatment parameters. Ultrasound propagation through a human skull fragment caused 44.4 ± 1% pressure attenuation at a normal incidence angle, while the focal size decreased by 3.3 ± 1.4% and 3.9 ± 1.8% along the lateral and axial dimension, respectively. Measured lateral and axial shifts were 0.5 ± 0.4 mm and 2.1 ± 1.1 mm, while simulated shifts were 0.1 ± 0.2 mm and 6.1 ± 2.4 mm, respectively. A 1.5-MHz passive cavitation detector transcranially detected cavitation signals of Definity microbubbles flowing through a vessel-mimicking phantom. T1-weighted MRI confirmed a 153 ± 5.5 mm3 BBB opening in two non-human primates at a mechanical index of 0.4, using Definity microbubbles at the FDA-approved dose for imaging applications, without edema or hemorrhage. In conclusion, we developed a portable system for non-invasive BBB opening in humans, which can be achieved at clinically relevant ultrasound exposures without the need for in-line MRI guidance. The proposed FUS system may accelerate the adoption of non-invasive FUS-mediated therapies due to its fast application, low cost and portability.

The blood-brain barrier (BBB) prevents most drugs from gaining access to the brain parenchyma, which is a recognized impediment to the treatment of neurodegenerative disorders like Parkinson's disease (PD). Focused ultrasound (FUS), in conjunction with systemically administered microbubbles, opens the BBB locally, reversibly and non-invasively. Herein, we show that neither FUS applied over both the striatum and the ventral midbrain, without neurotrophic factors, nor intravenous administration of neurotrophic factors (either through protein or gene delivery) without FUS, ameliorates the damage to the nigrostriatal dopaminergic pathway in the sub-acute MPTP mouse model of early-stage PD. Conversely, the combination of FUS and intravenous neurotrophic (protein or gene) delivery attenuates the damage to the nigrostriatal dopaminergic pathway, by allowing the entry of these agents into the brain parenchyma. Our findings provide evidence that the application of FUS at the early stages of PD facilitates critical neurotrophic delivery that can curb the rapid progression of neurodegeneration while improving the neuronal function, seemingly opening new therapeutic avenues for the early treatment of diseases of the central nervous system.

Objective. Focused ultrasound (FUS) has recently been demonstrated capable of exciting motor neuronal activity. However, comprehensive understanding of elucidated excitatory and inhibitory effects is required to better assess FUS-mediated modulation. In this study, we demonstrate that image-guided FUS can selectively modulate motor neuron activity in the mouse sciatic nerve in vivo and attribute motor responses to thermal effects. Approach. FUS was applied on the sciatic nerve of anesthetized mice in vivo through the intact skin and muscle using ultrasound imaging for targeting. Both excitatory and inhibitory effects were recorded using electromyography (EMG) along with muscle response of the hind limb. The effects of FUS modulation vs heating by invasive alternative heating source (AHS) on electrically evoked EMG responses in the sciatic nerve in vivo were also investigated. The safety and reversibility of the technique were validated using histology and EMG recovery. Main results. The FUS was capable of eliciting motor neuronal activity comparable to electrical stimulation, and facilitating motor neuronal response on electrically evoked potentials with temperature elevation up to 11.5 ± 0.3 oC (PRF ≤ 40 Hz). On the other hand, FUS-induced temperature elevations above 15.1 ± 1.6 oC (PRF ≥ 100 Hz) resulted in the suppression of electrically-evoked motor neuronal activity along with a decrease in EMG latency and area under the curve (AUC), which was validated against the invasive AHS with temperature elevation of 18.1 ± 8.5 oC instead of FUS modulation. Histological findings along with EMG responses after FUS modulation demonstrated a reversible or irreversible modulation. Significance. The findings reported herein indicate that image-guided FUS (PRF ≤ 100 Hz) induces safe and controllable modulation of involuntarily evoked motor neuron activity in vivo.

Conventional treatments of neuropathic pain are commonly invasive or non-localized. Focused ultrasound (FUS) is a promising neuromodulation technique that has been shown effective in various animal models. There have been numerous advances in the field of ultrasound neuromodulation with the technology already being applied to humans without a clear understanding of the underlying mechanism. Here, we present a method capable of targeting and monitoring of focused ultrasound (FUS) neuromodulation of the mouse sciatic nerve using high frame-rate displacement and cavitation imaging. Our technique is capable of detecting micron displacements and cavitation activity at the focus. Displacement and cavitation were measured with excitation of the sciatic nerve, indicating that radiation force and cavitation play in parts of the underlying mechanism. Taken together, our imaging technique is a powerful in vivo tool for real-time targeting of deep structures and investigation of the FUS neuromodulation mechanism.

Focused ultrasound (FUS) has demonstrated the ability to modulate nervous system activity in animals. Recently, higher frequency focused transducers have been used to successfully elicit motor responses in mice in vivo. Despite this success, quantitative analyses of the temporal and magnitude-related characteristics of FUS elicited motor responses measured with electromyography (EMG) and the spatial specificity of those responses have been poorly described. In this study, we use a focused transducer with high spatial resolution (1mm lateral focal diameter) and EMG to evaluate six quantitative EMG response characteristics. Results demonstrate that these responses are highly specific to the brain volume acted upon by the FUS

Brain diseases including neurological disorders and tumors remain under treated due to the challenge to access the brain, and blood-brain barrier (BBB) restricting drug delivery which, also profoundly limits the development of pharmacological treatment. Focused ultrasound (FUS) with microbubbles is the sole method to open the BBB noninvasively, locally, and transiently and facilitate drug delivery, while translation to the clinic is challenging due to long procedure, targeting limitations, or invasiveness of current systems. In order to provide rapid, flexible yet precise applications, we have designed a noninvasive FUS and monitoring system with the protocol tested in monkeys (from in silico preplanning and simulation, real-time targeting and acoustic mapping, to post-treatment assessment). With a short procedure (30 min) similar to current clinical imaging duration or radiation therapy, the achieved targeting (both cerebral cortex and subcortical structures) and monitoring accuracy was close to the predicted 2-mm lower limit. This system would enable rapid clinical transcranial FUS applications outside of the MRI system without a stereotactic frame, thereby benefiting patients especially in the elderly population.

Focused ultrasound (FUS) can noninvasively and reversibly modulate brain activity. Observed motor activity or behavioral changes are commonly used to infer neuromodulatory effects. Changes in autonomic nervous system regulation such as heart and respiratory function can potentially be used for studying the effects of FUS neuromodulation that do not produce outwardly observable motor or behavioral activity. In this study, low pressure (850 kPa), long duration (120 s) continuous-wave (CW) FUS at 2 MHz was targeted such that the focus spanned the visual cortex, hippocampus, and thalamus. These parameters were shown to reversibly depress heart rate in in vivo mice anesthetized with sodium pentobarbital by an average of 8.1 ± 3.7%. Following stimulus offset, heart rate either increased or varied in trend. Higher pressure (2 MPa), short duration (0.3 s) pulses at the same frequency resulted in either small, transient increases in heart rate or no change. Thermocouple measurements show the peak temperature elevation in the dura to be approximately 2 °C. Thermal simulations predict a peak temperature increase in the brain of 2.3 °C. These results indicate that CW FUS can alter autonomic nervous system regulation through very low heating and show that cardiorespiratory responses can be altered as a result of FUS neuromodulation.

Despite the progress of focused ultrasound (FUS)-mediated blood-brain barrier (BBB) disruption, neuro-inflammatory responses and the high variability of the FUS transmission through the human skull make the control of the acoustic parameters challenging. In this study, we developed a high-field (7-T) magnetic resonance (MR)-guided FUS system with a feedback control based on passive cavitation detection (PCD) to explore BBB opening in non-human primates (NHP). The sonication parameters were: 2 min duration, 500-kHz frequency, pulse length of 10 ms, and pulse repetition frequency of 5 Hz. T1-weighted MR images acquired every 5 min revealed a maximum contrast enhancement of 67% ± 15% relative to muscle after 30 min of sonication. Safe sonications were achieved in the 3 sessions using real-time PCD-based feedback control of the acoustic pressure. The high resolution anatomical images and the high temporal/spatial resolution of contrast agent diffusion provide a unique tool for studying the mechanisms of BBB disruption and drug delivery in NHP. Furthermore, the PCD-based feedback control allows repeatable safe sonication regardless of the variation of skull attenuation, allowing comparisons across animals and experimental sessions.

Embryonic and fetal mortality reduce reproductive performance of lactating dairy cows. The objectives of this study were to reduce pregnancy loss by administering a deslorelin implant (GnRH agonist) during the late embryonic period, to reduce follicular growth, induce accessory corpora lutea, and increase plasma progesterone concentrations. Lactating dairy cows received an implant containing 2.1 mg of deslorelin (Deslorelin group; n = 89) or no treatment (Control group; n = 92) on Day 27 of pregnancy. Pregnancy, ovarian structures and plasma progesterone concentrations were determined on Days 27 and 45, and pregnancy was re-confirmed on Day 90. On Day 45, mean +/- S.E.M. numbers of class 2 (6-9 mm; 0.72+/-0.19) and class 3 (or = 10 mm; 0.86 +/- 0.12) follicles for cows in the Deslorelin group were lower (P0.01) than the numbers of class 2 (1.90 +/- 0.18) and class 3 (1.92 +/- 0.12) follicles for cows in the Control group. On Day 45, the number of accessory corpora lutea for cows in the Deslorelin group (1.80 +/- 0.07) were greater (P0.01) than for cows in the Control group (1.31 +/- 0.07). On Day 45, plasma progesterone concentration was increased (P0.01) for cows in the Deslorelin group (8.03 +/- 0.33 ng/mL) compared to cows in the Control group (6.40 +/- 0.31 ng/mL). Pregnancy losses did not differ between Days 27 and 45 and Days 45 and 90 for cows in the Control (15.2 and 11.0%, respectively) and Deslorelin groups (20.2 and 10.5%, respectively). However, in the Deslorelin group, pregnancy loss between Days 45 and 90 was lower (P0.05) for cows that formed an accessory CL (0%) compared to cows that did not form an accessory CL (16.1%).

The hypercalcemia of various granulomatoses is caused by endogenous 1,25-dihydroxyvitamin D [1,25-(OH)2D3] overproduction by disease-activated macrophages. The inability of 1,25(OH)2D3 to suppress its synthesis in macrophages contrasts with the tight control of its production in macrophage precursors, peripheral blood monocytes (PBM). We examined whether 1,25(OH)2D3 resistance develops as PBM differentiate to macrophages or with macrophage activation. Normal human pulmonary alveolar macrophages (PAM) are less sensitive to 1,25(OH)2D3 than PBM, despite similar vitamin D receptor content; however, both PBM and PAM respond to exogenous 1,25-(OH)2D3 by inhibiting 1,25(OH)2D3 synthesis and inducing 1,25(OH)2D3 degradation through enhancement of 24-hydroxylase mRNA levels and activity. The human monocytic cell line THP-1 mimics PAM in 1,25(OH)2D3 synthesis and sensitivity to exogenous 1,25(OH)2D3. We utilized THP-1 cells to examine the response to 1,25(OH)2D3 with macrophage activation. Activation of THP-1 cells with gamma-interferon (gamma-IFN) enhances 1,25(OH)2D3 synthesis 30-fold, blocks 1,25-(OH)2D3 suppression of its synthesis, and reduces by 42.2% 1,25-(OH)2D3 induction of its degradation. The antagonistic effects of gamma-IFN are not merely restricted to enzymatic activities. In THP-1 cells and in normal PBM, gamma-IFN inhibits 1,25-(OH)2D3 induction of 24-hydroxylase mRNA levels without reducing mRNA stability, suggesting gamma-IFN inhibition of 1,25(OH)2D3 transactivating function. These results explain 1,25(OH)2D3 overproduction in granulomatoses and demonstrate potent inhibition by gamma-IFN of 1,25(OH)2D3 action in immune cells.

In chronic uremia, the requirement of supraphysiological doses of serum 25-hydroxyvitamin D3 [25(OH)D3] for the normalization of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels has been attributed to impaired substrate availability to renal 1 alpha-hydroxylase. Because serum 1,25(OH)2D3 can also be corrected by 25(OH)D3 supplementation in bilaterally nephrectomized patients, we examined the role of substrate availability on 1,25(OH)2D3 production by peripheral blood monocytes (PBM). In hemodialysis patients (HP), 25(OH)D3 uptake was 50% lower than normal, and the maximal velocity (Vmax) and apparent Michaelis constant (Km) for 25(OH)D3 of 1 alpha-hydroxylase were 2.7- and 4-fold above normal, respectively. When serum 1,25(OH)2D3 of HP was corrected by intravenous 1,25(OH)2D3, 25(OH)D3 uptake, Km, and Vmax returned to normal values. The effect of 25(OH)D3 supplementation was also examined. In normal adults, 25(OH)D3 administration had no effect on serum 1,25(OH)2D3 levels nor on the Km or the Vmax of PBM 1 alpha-hydroxylase but caused a 11-fold increase in serum 24R,25-dihydroxyvitamin D3[24R, 25(OH)2D3]. In HP, 25(OH)D3 therapy raised serum 1,25(OH)2D3 and reduced the Km and Vmax of PBM 1 alpha-hydroxylase, which correlated negatively with serum 1,25(OH)2D3. However, serum 24R,25(OH)2D3 only increased slightly above basal. These results demonstrate that, in HP, 1) impaired uptake of 25(OH)D3 and low affinity for substrate determine the need for high 25(OH)D3 levels to normalize serum 1,25(OH)2D3, despite higher enzymatic activity; 2) 1,25(OH)2D3 deficiency plays a role in enhanced 1,25(OH)2D3 synthesis and impaired access of 25(OH)D3 to PBM 1 alpha hydroxylase; and 3) abnormal 25(OH)D3 delivery also affects 24-hydroxylation.

An 8-month-old Japanese Black heifer with severe erythropoietic symptoms was subjected to clinical, histological and cytological examinations. During the 1 month clinical observation period, severe increases in RBC count, packed cell volume and haemoglobin concentration were observed. The plasma erythropoietin (Epo) concentration of the heifer (20.7 mIU/ml) was similar to that observed in normal control heifers. Blood gas examinations of the arterial and venous blood revealed low levels of partial pressure O(2) (PaO(2)), partial pressure CO(2) (PaCO(2)) and O(2) saturation (SaO(2)), while the blood pH was within the normal range. Gross lesions could not be detected. However, microscopic observation revealed severe proliferation of erythroblasts in the bone marrow and in the spleen without evidence of neoplastic changes. Based on these clinical and pathological examinations, we diagnosed the heifer as being the first case of primary erythrocytosis in Japanese Black cattle.

A serious decline in the reproductive performance of dairy cows occurs in southern Japan in the summer period, when the total number of hot days ≥35°C numbers more than 20 days annually. Previous reports have mentioned the effectiveness of embryo transfer (ET) at 7 days after AI (AI/ET) under heat-stressed conditions. In the present study, we investigated the effect of AI/ET on conception rate (CR) under heat-stressed conditions in the summer period. Artificial insemination was performed at 13 commercial dairies in this study from August through September in 2007 and 2008. Seven days after AI, a single embryo was transferred into the uterine horn contralateral to the ovary with a corpus luteum (AI/ET, n = 82). Artificial insemination at oestrus without further treatment was assigned as the control group (AI, n = 367). In 2007, frozen–thawed embryos of Japanese Black cattle were transferred, and the same cattle were used for ET of fresh embryos in 2008. The temperature-humidity index [0.8 × temperature + 0.01 ×relative humidity (temperature –14.4) + 46.4], rectal temperature, and diurnal highest or lowest and average ambient temperatures were measured at the time of AI and ET. Cows were diagnosed for pregnancy at 42 days after AI by palpation per rectum and were reexamined by transrectal ultrasonography at 60 days after AI. The CR was calculated as the number of cows diagnosed as pregnant 60 days after AI divided by the number of cows inseminated. Fetal loss was calculated as the number of cows that did not deliver calves after term divided by the number of cows diagnosed as pregnant. The CR, number of AI, fetal loss, and type of newborn (Holsteins, AI origin; Japanese Black, ET origin) were confirmed retrospectively. For statistical analysis, Fisher’s exact test and Student’s t-test were used for comparison of the CR, fetal loss, and body temperature by using a statistical software program for PC (Excel Statistics 2006). The CR for AI/ET was 30.4% and for AI was 13.8% in 2007 (P 0.05). The fetal loss was 38.1% in AI/ET v. 7.4% in AI in 2007 (P

Oxacalcitriol (1,25-(OH)Z-220xa-D3) mimics the action of 1,25- dihydroxyvitamin D3 (1,25-(OH),D,) in a variety of target tissues, including the systemic control of calcitriol metabolism. Similar to 1,25- (OH)1D3, 1,25-(OH)Z-220xa-D3 decreases the rate of 1,25-(OH)2D3 syn- thesis and accelerates its metabolic clearance rate. We have previously shown that in normal human monocytes, physiological concentrations of 1,25-(OH)*D3 and 1,25-(OH)2-220xa-D3 determine identical suppres- sion of 1,25-(OH)*D3 synthesis. Moreover, both sterols have a similar potency to induce vitamin D degradation through stimulation of the C24-hydroxylation pathway. In this study, we examined the ability of normal human monocytes to metabolize 1,25-(OH)2-220xa-D3 and whether the enzymes involved are the same as those that catabolize 1,25-(OH)2D3. Time-course experiments demonstrated no detectable basal catabolic activity. However, exogenous 1,25-(OH)QDa at physio- logical concentrations induced 1,25-(OH)2-220xa-D3 degradation by normal human monocytes. Competition experiments showed that a lo- fold molar excess of unlabeled 1,25-(OH)*D3 inhibited tritiated-1,25- (OH)2-220xa-D3 catabolism by 85%, whereas a lo-fold excess of unla- beled 1,25-(OH)2-220xa-D3 reduced tritiated-1,25-(OH)2-220xa-D3 ca- tabolism by 33%. In contrast, although a lo-fold excess of unlabeled 1,25-(OH)2D3 reduced tritiat=ed 1,25-(OH)2D3 catabolism by 60%, a 1000-fold excess of 1,25-(OH)2-220xa-D3 was required to reduce triti- ated 1,25-(OH)*D3 catabolism to this degree. The apparent K, for 1,25- (OH)2-220xa-D3 was significantly higher than that of 1,25-(OH)*D3 (2.0 rt 0.8 us. 0.9 -t 0.2 nM, respectively; P < 0.001) for the catabolic pathway induced by physiological concentrations of 1,25-(OH)2D3. Moreover, the presence of 0.65 nM lr25-(OH)ZD3 caused an additional increase in the K, for 1,25-(OH)Z-220xa-D3 (3.2 rf: 0.8 nM). These data suggest that 1,25-(OH)z-220xa-D3 may be less accessible than 1,25-(OH)zD3 to the hydroxylases involved in vitamin D catabolism. The resulting prolonged biological half-life of the analog in certain target tissues may be involved in its selectivity. (Endocrinology 133: 2719-2723, 1993)