Wendy Oakden

Microbubbles loaded with nanoparticles: a route to multiple imaging modalities.

We report a single-step approach to producing small and stable bubbles functionalized with nanoparticles. The strategy includes the following events occurring in sequence: (i) a microfluidic generation of bubbles from a mixture of CO(2) and a minute amount of gases with low solubility in water, in an aqueous solution of a protein, a polysaccharide, and anionic nanoparticles; (ii) rapid dissolution of CO(2) leading to the shrinkage of bubbles and an increase in acidity of the medium in the vicinity of the bubbles; and (iii) co-deposition of the biopolymers and nanoparticles at the bubble-liquid interface. The proposed approach yielded microbubbles with a narrow size distribution, long-term stability, and multiple functions originating from the attachment of metal oxide, metal, or semiconductor nanoparticles onto the bubble surface. We show the potential applications of these bubbles in ultrasound and magnetic resonance imaging.

Observation of nonlinear shear wave propagation using magnetic resonance elastography

MR elastography (MRE) is an MRI modality that is increasingly being used to image tissue elasticity throughout the body. One MRE technique that has received a great deal of attention is based on visualizing shear waves, which reveal stiffness by virtue of their local wavelength. However, the shape of propagating shear waves can also provide valuable information about the nonlinear stress–strain behavior of tissue. Here an experiment is proposed that allows the observation of nonlinear wave propagation based on spatial‐temporal phase contrast images. A theoretical description of the wave propagation was developed that reflects typical MRE excitation, which involves excitation modes both parallel and perpendicular to B0. Based on this model, it is shown that both odd and even higher harmonics are produced with their amplitudes dependent on the details of the actuator, imaging geometry, and the nonlinear tissue properties. With appropriate motion encoding, harmonic vibrations arising from nonlinear tissue response can be detected. The effect is demonstrated on an agarose gel phantom using a sinusoidal shear vibration of 150 Hz, and clearly shows the presence of harmonics at 600 and 750 Hz. Using an estimate of the strain energy of the phantom, we were able to determine the nonlinear tissue properties. Magn Reson Med 52:842–850, 2004.

Gene delivery to the spinal cord using MRI-guided focused ultrasound

Non-invasive gene delivery across the blood–spinal cord barrier (BSCB) remains a challenge for treatment of spinal cord injury and disease. Here, we demonstrate the use of magnetic resonance image-guided focused ultrasound (MRIgFUS) to mediate non-surgical gene delivery to the spinal cord using self-complementary adeno-associated virus serotype 9 (scAAV9). scAAV9 encoding green fluorescent protein (GFP) was injected intravenously in rats at three dosages: 4 × 108, 2 × 109 and 7 × 109 vector genomes per gram (VG g−1). MRIgFUS allowed for transient, targeted permeabilization of the BSCB through the interaction of focused ultrasound (FUS) with systemically injected Definity lipid-shelled microbubbles. Viral delivery at 2 × 109 and 7 × 109 VG g−1 leads to robust GFP expression in FUS-targeted regions of the spinal cord. At a dose of 2 × 109 VG g−1, GFP expression was found in 36% of oligodendrocytes, and in 87% of neurons in FUS-treated areas. FUS applications to the spinal cord could address a long-term goal of gene therapy: delivering vectors from the circulation to diseased areas in a non-invasive manner.

Closure of the annulus fibrosus of the intervertebral disc using a novel suture application device—in vivo porcine and ex vivo biomechanical evaluation

BACKGROUND CONTEXT Defects in the annulus fibrosus (AF) remain a challenge in the surgical treatment of lumbar disc herniations with persistent defects, allowing potential re herniation of nucleus pulposus (NP) tissue. A cervical porcine model was chosen to simulate human lumbar intervertebral disc (IVD). PURPOSE The aim of this study was to determine the technical feasibility of closure of the AF of the IVD using a novel minimally invasive Kerrison-shaped suture application device. STUDY DESIGN Ex vivo biomechanical and in vivo porcine device evaluations were performed. METHODS Ex vivo biomechanical evaluation: 15 porcine spinal units were explanted and subjected to mock discectomy. The annular defect was closed using 2-0 non-absorbable (ultra-high molecular-weight polyethylene, UHMWPE) suture and Dines knot. The knot was backed up with two, three, or four throws. The spinal unit was subject to 4000 cycles of flexion/extension with 1500 N of axial load, and assessed for knot slippage. In vivo porcine device evaluation: three pigs (53-57 kg) were anesthetized and underwent a ventral surgical approach to the cervical spine. The AF of two discs was incised, and simulated partial NP discectomy was performed. The defect was closed at one level using the AnchorKnot device to apply the suture with a Dines knot and four throws. The pigs were observed for 4 weeks before euthanasia, allowing 7T magnetic resonance imaging (MRI) and histological evaluation. RESULTS A Dines knot with four throws experienced no slippage after 4000 cycles. This configuration was tested in vivo. Clinically, the neurological examination in treated pigs was normal following surgery. Histological and MRI assessment confirmed sustained defect closure at 4 weeks. There was no reaction to the suture material and no NP extrusion at any of the sutured levels. CONCLUSIONS This study demonstrates that it is technically feasible to perform AF defect closure in a porcine model. This novel device achieved AF defect closure that was maintained through 4 weeks in vivo.

Prolonged Subdural Infusion of Kynurenic Acid Is Associated with Dose-Dependent Myelin Damage in the Rat Spinal Cord.

Background Kynurenic acid (KYNA) is the end stage metabolite of tryptophan produced mainly by astrocytes in the central nervous system (CNS). It has neuroprotective activities but can be elevated in the neuropsychiatric disorders. Toxic effects of KYNA in the CNS are unknown. The aim of this study was to assess the effect of the subdural KYNA infusion on the spinal cord in adult rats. Methods A total of 42 healthy adult rats were randomly assigned into six groups and were infused for 7 days with PBS (control) or 0.0002 pmol/min, 0.01 nmol/min, 0.1 nmol/min, 1 nmol/min, and 10 nmol/min of KYNA per 7 days. The effect of KYNA on spinal cord was determined using histological and electron microscopy examination. Myelin oligodendrocyte glycoprotein (MOG) was measured in the blood serum to assess a degree of myelin damage. Result In all rats continuous long-lasting subdural KYNA infusion was associated with myelin damage and myelin loss that was increasingly widespread in a dose-depended fashion in peripheral, sub-pial areas. Damage to myelin sheaths was uniquely related to the separation of lamellae at the intraperiod line. The damaged myelin sheaths and areas with complete loss of myelin were associated with limited loss of scattered axons while vast majority of axons in affected areas were morphologically intact. The myelin loss-causing effect of KYNA occurred with no necrosis of oligodendrocytes, with locally severe astrogliosis and no cellular inflammatory response. Additionally, subdural KYNA infusion increased blood MOG concentration. Moreover, the rats infused with the highest doses of KYNA (1 and 10 nmol/min) demonstrated adverse neurological signs including weakness and quadriplegia. Conclusions We suggest, that subdural infusion of high dose of KYNA can be used as an experimental tool for the study of mechanisms of myelin damage and regeneration. On the other hand, the administration of low, physiologically relevant doses of KYNA may help to discover the role of KYNA in control of physiological myelination process.

Effects of diffusion on high-resolution quantitative T2 MRI.

Carr–Purcell–Meiboom–Gill‐based sequences are often assumed to be insensitive to diffusion. However, imaging gradients always contribute some degree of diffusion weighting which increases with resolution. This may cause an apparent decrease in T2 when using a multi‐echo sequence, such as quantitative T2 (qT2) at high resolution. This study investigated the impact of diffusion on the qT2 sequence. An equation was developed relating the diffusion factor associated with each echo (bqT2) to the underestimation of T2, which was strongly dependent on both the actual T2 and the apparent diffusion coefficient of the tissue. The diffusion dependence of the measured T2 was demonstrated in rat spinal cord. The measured T2 was independent of the imaging plane in gray matter, where diffusion was isotropic, and orientation dependent in white matter, where diffusion was strongly anisotropic. The dependence of the measured T2 on the actual T2 value was also demonstrated in MnCl2 phantoms. The relationship between the resolution and underestimation of T2 was investigated both theoretically and experimentally for the original readout and a fully refocused readout. The fully refocused readout increased the resolution at which diffusion effects could be neglected whilst measuring T2. To avoid the misidentification of cerebrospinal fluid when applying qT2 in the brain or spinal cord, a minimum in‐plane voxel dimension of 0.2 mm was suggested for the standard qT2 sequence and 0.1 mm for the refocused readout. Simulations of myelin water fraction measurement indicated that signal‐to‐noise ratio requirements were increased in the presence of diffusion. Finally, the use of decreasing spoiler gradients to attenuate stimulated echoes should be avoided, as it was found to distort the T2 distribution when the slice thickness was less than 1 mm. Copyright

An in vivo model of anti-inflammatory activity of subdural dexamethasone following the spinal cord injury

Current therapies to limit the neural tissue destruction following the spinal cord injury are not effective. Our recent studies indicate that the injury to the white matter of the spinal cord results in a severe inflammatory response where macrophages phagocytize damaged myelin and the fluid-filled cavity of injury extends in size with concurrent and irreversible destruction of the surrounding neural tissue over several months. We previously established that a high dose of 4mg/rat of dexamethasone administered for 1 week via subdural infusion remarkably lowers the numbers of infiltrating macrophages leaving large amounts of un-phagocytized myelin debris and therefore inhibits the severity of inflammation and related tissue destruction. But this dose was potently toxic to the rats. In the present study the lower doses of dexamethasone, 0.125-2.0mg, were administered via the subdural infusion for 2 weeks after an epidural balloon crush of the mid-thoracic spinal cord. The spinal cord cross-sections were analyzed histologically. Levels of dexamethasone used in the current study had no systemic toxic effect and limited phagocytosis of myelin debris by macrophages in the lesion cavity. The subdural infusion with 0.125-2.0mg dexamethasone over 2 week period did not eliminate the inflammatory process indicating the need for a longer period of infusion to do so. However, this treatment has probably lead to inhibition of the tissue destruction by the severe, prolonged inflammatory process.

A non-surgical model of cervical spinal cord injury induced with focused ultrasound and microbubbles.

BACKGROUND The most commonly used animal models of spinal cord injury (SCI) involve surgical exposure of the dorsal spinal cord followed by transection, contusion or compression. This high level of invasiveness often requires significant post-operative care and can limit post-operative imaging, as the surgical incision site can interfere with coil placement for magnetic resonance imaging (MRI) during the acute phase of SCI. While these models are considered to be similar to human SCI, they do not occur in a closed vertebral system as do the majority of human injuries. NEW METHOD Here we describe a novel, non-surgical model of SCI in the rat using MR-guided focused ultrasound (FUS) in combination with intravenous injection of microbubbles, applied to the cervical spinal cord. RESULTS The injury was well-tolerated and resulted in cervical spinal cord damage in 60% of the animals. The area of Gd-enhancement immediately post-FUS and area of signal abnormality at 24h were correlated with the degree of injury. The extent of injury was easily visualized with T2-weighted MRI and was confirmed using histology. COMPARISON WITH EXISTING METHOD(S) Pathology was similar to that seen in other rat models of direct spinal cord contusion and compression. Unlike these methods, FUS is non-surgical and has lower mortality than seen in other models of cervical SCI. CONCLUSIONS We developed a novel model of SCI which was non-surgical, well-tolerated, localized, and replicated the pathology seen in other models of SCI.

Quantitative MRI in a non-surgical model of cervical spinal cord injury

Quantitative T2 (qT2), diffusion tensor imaging (DTI), and histology were used to investigate a cervical model of spinal cord injury (SCI) in the rat. While quantitative MRI can significantly increase the specificity in the presence of pathology, it must be validated for each type of injury or disease. In the case of traumatic SCI most models are difficult to image, either due to the location of the injury, or as a result of damage to surrounding tissues resulting from invasive surgical procedures. In this study a non‐surgical cervical model of SCI, produced using a combination of focused ultrasound and microbubbles, was used to produce pathology similar to that seen in models of contusive and compressive injuries. qT2 and DTI were performed at 24 h and 1 and 2 weeks following injury, and compared with H&E and luxol fast blue histology. In the injured spinal cord, in addition to intra/extracellular (I/E) water and myelin water in white matter, qT2 revealed a large component with very short T2 of about 3 ms, which was highly correlated with the presence of hemorrhage in both gray and white matter at 24 h, and with the presence of hemosiderin in gray matter at 2 weeks following injury. The T2 of the I/E water peak was also elevated at 24 h in both gray and white matter, which was correlated with the presence of vacuolation/edema on histology. Cystic cavities were only seen at the 1 or 2 week timepoints, and were correlated with the presence of a water peak with T2 > 250 ms. No significant changes in diffusivity parameters were observed. Pathologies were often co‐occurring, with opposite effects on the average T2 in a given voxel, reducing the visibility of injured tissue on standard T2‐weighted MR images. Copyright

Differences in iron and manganese concentration may confound the measurement of myelin from R1 and R2 relaxation rates in studies of dysmyelination

A model of dysmyelination, the Long Evans Shaker (les) rat, was used to study the contribution of myelin to MR tissue properties in white matter. A large region of white matter was identified in the deep cerebellum and was used for measurements of the MR relaxation rate constants, R1 = 1/T1 and R2 = 1/T2, at 7 T. In this study, R1 of the les deep cerebellar white matter was found to be 0.55 ± 0.08 s –1 and R2 was found to be 15 ± 1 s–1, revealing significantly lower R1 and R2 in les white matter relative to wild‐type (wt: R1 = 0.69 ± 0.05 s–1 and R2 = 18 ± 1 s–1). These deviated from the expected ΔR1 and ΔR2 values, given a complete lack of myelin in the les white matter, derived from the literature using values of myelin relaxivity, and we suspect that metals could play a significant role. The absolute concentrations of the paramagnetic transition metals iron (Fe) and manganese (Mn) were measured by a micro‐synchrotron radiation X‐ray fluorescence (μSRXRF) technique, with significantly greater Fe and Mn in les white matter than in wt (in units of μg [metal]/g [wet weight tissue]: les: Fe concentration,19 ± 1; Mn concentration, 0.71 ± 0.04; wt: Fe concentration,10 ± 1; Mn concentration, 0.47 ± 0.04). These changes in Fe and Mn could explain the deviations in R1 and R2 from the expected values in white matter. Although it was found that the influence of myelin still dominates R1 and R2 in wt rats, there were non‐negligible changes in the contribution of the metals to relaxation. Although there are already problems with the estimation of myelin from R1 and R2 changes in disease models with pathology that also affects the relaxation rate constants, this study points to a specific pitfall in the estimation of changes in myelin in diseases or models with disrupted concentrations of paramagnetic transition metals. Copyright