Functional neuroimaging using ultrasonic blood-brain barrier disruption and manganese-enhanced MRI.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents. In AIM MRI, Mn(2+) acts a calcium analog and accumulates in depolarized neurons. Because Mn(2+) shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn(2+) clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn(2+) does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice. One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage. Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response. To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

Contrast-enhanced in vivo magnetic resonance microscopy of the mouse brain enabled by noninvasive opening of the blood-brain barrier with ultrasound.

The use of contrast agents for neuroimaging is limited by the blood-brain barrier (BBB), which restricts entry into the brain. To administer imaging agents to the brain of rats, intracarotid infusions of hypertonic mannitol have been used to open the BBB. However, this technically challenging approach is invasive, opens only a limited region of the BBB, and is difficult to extend to mice. In this work, the BBB was opened in mice, using unfocused ultrasound combined with an injection of microbubbles. This technique has several notable features: it (a) can be performed transcranially in mice; (b) takes only 3 min and uses only commercially available components; (c) opens the BBB throughout the brain; (d) causes no observed histologic damage or changes in behavior (with peak-negative acoustic pressures of 0.36 MPa); and (e) allows recovery of the BBB within 4 h. Using this technique, Gadopentetate Dimeglumine (Gd-DTPA) was administered to the mouse brain parenchyma, thereby shortening T(1) and enabling the acquisition of high-resolution (52 × 52 × 100 micrometers(3)) images in 51 min in vivo. By enabling the administration of both existing anatomic contrast agents and the newer molecular/sensing contrast agents, this technique may be useful for the study of mouse models of neurologic function and pathology with MRI.

Ultrasonic disruption of the blood-brain barrier enables in vivo functional mapping of the mouse barrel field cortex with manganese-enhanced MRI.

Though mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents. In AIM MRI, manganese(2+) acts a calcium analog and accumulates in depolarized neurons. Because manganese(2+) shortens T1, regions of elevated neuronal activity enhance in MRI. However, because manganese does not cross the blood-brain barrier (BBB), the need to osmotically disrupt the BBB has limited the use of AIM MRI, particularly in mice. In this work, the BBB was opened in mice using unfocused, transcranial ultrasound in combination with gas-filled microbubbles. Using this noninvasive technique to open the BBB bilaterally, manganese could be quickly administered to the whole mouse brain. With this approach, AIM MRI was used to map the neuronal response to unilateral mechanical stimulation of the vibrissae in lightly sedated mice. The resultant 3D activation map agreed well with published representations of the vibrissae regions of the barrel field cortex. The anterior portions of the barrel field cortex corresponding to the more rostral vibrissae showed greater activation, consistent with previous literature. Because the ultrasonic opening of the BBB is simple, fast, and noninvasive, this approach is suitable for high-throughput and longitudinal studies in awake mice. This approach enables a new way to map neuronal activity in mice with manganese.

High-resolution magnetic resonance angiography in the mouse using a nanoparticle blood-pool contrast agent.

High-resolution magnetic resonance angiography is already a useful tool for studying mouse models of human disease. Magnetic resonance angiography in the mouse is typically performed using time-of-flight contrast. In this work, a new long-circulating blood-pool contrast agent-a liposomal nanoparticle with surface-conjugated gadolinium (SC-Gd liposomes)-was evaluated for use in mouse neurovascular magnetic resonance angiography. A total of 12 mice were imaged. Scan parameters were optimized for both time-of-flight and SC-Gd contrast. Compared to time-of-flight contrast, SC-Gd liposomes (0.08 mmol/kg) enabled improved small-vessel contrast-to-noise ratio, larger field of view, shorter scan time, and imaging of venous structures. For a limited field of view, time-of-flight and SC-Gd were not significantly different; however, SC-Gd provided better contrast-to-noise ratio when the field of view encompassed the whole brain (P < 0.001) or the whole neurovascular axis (P < 0.001). SC-Gd allowed acquisition of high-resolution magnetic resonance angiography (52 x 52 x 100 micrometer(3) or 0.27 nL), with 123% higher (P < 0.001) contrast-to-noise ratio in comparable scan time ( approximately 45 min). Alternatively, SC-Gd liposomes could be used to acquire high-resolution magnetic resonance angiography (0.27 nL) with 32% higher contrast-to-noise ratio (P < 0.001) in 75% shorter scan time (12 min).

Rapid production of specialized animal handling devices using computer-aided design and solid freeform fabrication.

PURPOSE: To develop a process for rapidly and inexpensively producing customized animal handling devices for small animal imaging. MATERIALS AND METHODS: To meet the specific needs of a particular imaging experiment, measurements are taken from imaging data and the animal handling devices are designed using 3D computer-aided design (CAD) software. Parts are produced in a few days using solid freeform fabrication (SFF, a.k.a. rapid prototyping). RESULTS: This process is illustrated with the production of an animal handling system for stereotaxically prescribed therapeutic ultrasound and MRI of the mouse brain. The device provides integrated head-fixation, anesthesia delivery, and physiological monitoring in a modular system. Design and production took approximately 1 week and the cost was a small fraction of a traditional machine shop. CONCLUSION: Commercial animal handling products typically have limited functionality and are not integrated with other laboratory infrastructure. However, using CAD and SFF, sophisticated animal handling devices can be produced to meet the specific experimental needs. This process is typically faster and less expensive than using a traditional machine shop, and the products are more robust than typical homemade devices. Using high-quality purpose-built devices permits experiments to be executed with greater consistency and higher throughput.

Blood-brain barrier (BBB) disruption using a diagnostic ultrasound scanner and Definity in Mice.

The objective of this work was to determine whether diagnostic ultrasound and contrast agent could be used to transcranially and nondestructively disrupt the blood-brain barrier (BBB) in mice under ultrasound image guidance and to quantify that disruption using magnetic resonance imaging (MRI) and magnetic resonance (MR) contrast agent. Each mouse was placed under isoflurane anesthesia and the hair on top of its skull was removed before treatment. A diagnostic ultrasound transducer was placed in a water bag coupled with gel on the mouse skull. Definity (ultrasound [US] contrast) and Magnevist (MR contrast) were injected concurrent with the start of a custom ultrasound transmission sequence. The transducer was translated along the rostral-caudal axis to insonify three spatial locations (2mm apart) along one half of the brain for each sequence. T1-weighted MR images were used to quantify the volume of tissue over which the BBB disruption allowed Magnevist to enter the brain, based upon increases in MR contrast-to-noise ratio (CNR) compared with the noninsonified portions of the brain. Ultrasonic frequency, pressure and pulse duration, as well as Definity dose and injection time were varied. Preliminary results suggest that a threshold exists for BBB opening dependent upon both pressure and pulse duration (consistent with reports in the literature performed at lower frequencies). A range of typical diagnostic frequencies (e.g., 5.0-8.0 MHz) generated BBB disruption. Comparable BBB opening was noted with varied delays between Definity injection and insonification (0-2 min) for a range of Definity concentrations (400-2400 microL/kg). The low-pressure, custom sequences (mechanical index [MI]< or =0.65) had minimal blood cell extravasation as determined by histologic evaluation. This study has shown the ability of a diagnostic ultrasound system, in conjunction with Definity, to open the BBB transcranially in a mouse model for molecules approximately 0.5 kDa in size. Opening was achieved at higher frequencies than previously reported and was localized under ultrasound image guidance. A typical, ultrasound imaging mode (pulsed wave [PW] Doppler) with specific settings (transmit frequency=5.7 MHz, gate size=15 mm, pulse repetition frequency=100 Hz, system power=15%) successfully opened the BBB, which facilitates implementation using the most of commercially available clinical diagnostic scanners. Localized opening of the BBB may have potential clinical utility for the delivery of diagnostic or therapeutic agents to the brain.

Genetic regulation of endotoxin-induced airway disease.

To identify novel genes regulating the biologic response to lipopolysaccharide (LPS), we used a combination of quantitative trait locus (QTL) analysis and microarray-based gene expression studies of C57BL/6J x DBA/2J(BXD) F2 and recombinant inbred (RI) mice. A QTL affecting pulmonary TNF-alpha production was identified on chromosome 2, and a region affecting both polymorphonuclear leukocyte recruitment and TNF-alpha levels was identified on chromosome 11. Microarray analyses of unchallenged and LPS-challenged BXD RI strains identified approximately 500 genes whose expression was significantly changed by inhalation of LPS. Of these genes, 28 reside within the chromosomal regions identified by the QTL analyses, implicating these genes as high priority candidates for functional studies. Additional high priority candidate genes were identified based on their differential expression in mice having high and low responses to LPS. Functional studies of these genes are expected to reveal important molecular mechanisms regulating the magnitude of biologic responses to LPS.