Contrast-enhanced ultrasound imaging detects anatomical and functional changes in rat cervical spine microvasculature with normal aging.

Normal aging is associated with significant deleterious cerebrovascular changes; these have been implicated in disease pathogenesis and increased susceptibility to ischemic injury. While these changes are well documented in the brain, few studies have been conducted in the spinal cord. Here, we utilize specialized contrast-enhanced ultrasound (CEUS) imaging to investigate age-related changes in cervical spinal vascular anatomy and hemodynamics in male Fisher 344 rats, a common strain in aging research. Aged rats (24-26 mo., N=6) exhibited significant tortuosity in the anterior spinal artery and elevated vascular resistance compared to adults (4-6 mo., N=6; tortuosity index 2.20±0.15 vs 4.74±0.45, p<0.05). Baseline blood volume was lower in both larger vessels and the microcirculation in the aged cohort, specifically in white matter (4.44e14±1.37e13 vs 3.66e14±2.64e13 CEUS bolus AUC, p<0.05). To elucidate functional differences, animals were exposed to a hypoxia challenge; whereas adult rats exhibited significant functional hyperemia in both gray and white matter (GM: 1.13±0.10-fold change from normoxia, p<0.05; WM: 1.16±0.13, p<0.05), aged rats showed no response. Immunohistochemistry revealed reduced pericyte coverage and activated microglia behavior in aged rats, which may partially explain the lack of vascular response. This study provides the first in vivo description of age-related hemodynamic differences in the cervical spinal cord.

Quantifying injury expansion in the cervical spinal cord with intravital ultrafast contrast-enhanced ultrasound imaging.

Spinal cord injury is characterized by hemodynamic disruption at the injury epicenter and hypoperfusion in the penumbra, resulting in progressive ischemia and cell death. This degenerative secondary injury process has been well-described, though mostly using ex vivo or depth-limited optical imaging techniques. Intravital contrast-enhanced ultrasound enables longitudinal, quantitative evaluation of anatomical and hemodynamic changes in vivo through the entire spinal parenchyma. Here, we used ultrasound imaging to visualize and quantify subacute injury expansion (through 72 h post-injury) in a rodent cervical contusion model. Significant intraparenchymal hematoma expansion was observed through 72 h post-injury (1.86 ± 0.17-fold change from acute, p < 0.05), while the volume of the ischemic deficit largely increased within 24 h post-injury (2.24 ± 0.27-fold, p < 0.05). Histology corroborated these findings; increased apoptosis, tissue and vessel loss, and sustained tissue hypoxia were observed at 72 h post-injury. Vascular resistance was significantly elevated in the remaining perfused tissue, likely due in part to deformation of the central sulcal artery nearest to the lesion site. In conjunction, substantial hyperemia was observed in all perilesional areas examined except the ipsilesional gray matter. This study demonstrates the utility of longitudinal ultrasound imaging as a quantitative tool for tracking injury progression in vivo.

A decrease in the neuroprotective effects of acute spinal cord decompression according to injury severity: introducing the concept of a ceiling effect.

OBJECTIVE: Acute traumatic spinal cord injury (tSCI) is followed by a prolonged period of secondary neuroglial cell death. Neuroprotective interventions, such as surgical spinal cord decompression, aim to mitigate secondary injury. In this study, the authors explore whether the effect size of posttraumatic neuroprotective spinal cord decompression varies with injury severity. METHODS: Seventy-one adult female Long Evans rats were subjected to a thoracic tSCI using a third-generation spinal contusion device. Moderate and severe tSCI were defined by recorded impact force delivered to the spinal cord. Immediately after injury (< 15 minutes), treatment cohorts underwent either a decompressive durotomy or myelotomy. Functional recovery was documented using the Basso, Beattie, and Bresnahan locomotor scale, and tissue sparing was documented using histological analysis. RESULTS: Moderate and severe injuries were separated at a cutoff point of 231.8 kdyn peak impact force based on locomotor recovery at 8 weeks after injury. Durotomy improved hindlimb locomotor recovery 8 weeks after moderate trauma (p < 0.01), but not after severe trauma (p > 0.05). Myelotomy led to increased tissue sparing (p < 0.0001) and a significantly higher number of spared motor neurons (p < 0.05) in moderate trauma, but no such effect was noted in severely injured rats (p > 0.05). Within the moderate injury group, myelotomy also resulted in significantly more spared tissue when compared with durotomy-only animals (p < 0.01). CONCLUSIONS: These results suggest that the neuroprotective effects of surgical spinal cord decompression decrease with increasing injury severity in a rodent tSCI model.

Quantitative tissue perfusion imaging using nonlinear ultrasound localization microscopy.

Ultrasound localization microscopy (ULM) is a recent advancement in ultrasound imaging that uses microbubble contrast agents to yield vascular images that break the classical diffraction limit on spatial resolution. Current approaches cannot image blood flow at the tissue perfusion level since they rely solely on differences in velocity to separate tissue and microbubble signals; lower velocity microbubble echoes are removed during high pass wall filtering. To visualize blood flow in the entire vascular tree, we have developed nonlinear ULM, which combines nonlinear pulsing sequences with plane-wave imaging to segment microbubble signals independent of their velocity. Bubble localization and inter-frame tracking produces super-resolved images and, with parameters derived from the bubble tracks, a rich quantitative feature set that can describe the relative quality of microcirculatory flow. Using the rat spinal cord as a model system, we showed that nonlinear ULM better resolves some smaller branching vasculature compared to conventional ULM. Following contusion injury, both gold-standard histological techniques and nonlinear ULM depicted reduced in-plane vessel length between the penumbra and contralateral gray matter (-16.7% vs. -20.5%, respectively). Here, we demonstrate that nonlinear ULM uniquely enables investigation and potential quantification of tissue perfusion, arguably the most important component of blood flow.

Early Detrusor Application of Botulinum Toxin A Results in Reduced Bladder Hypertrophy and Fibrosis after Spinal Cord Injury in a Rodent Model.

Following spinal cord injury (SCI), pathological reflexes develop that result in altered bladder function and sphincter dis-coordination, with accompanying changes in the detrusor. Bladder chemodenervation is known to ablate the pathological reflexes, but the resultant effects on the bladder tissue are poorly defined. In a rodent model of contusion SCI, we examined the effect of early bladder chemodenervation with botulinum toxin A (BoNT-A) on bladder histopathology and collagen deposition. Adult female Long Evans rats were given a severe contusion SCI at spinal level T9. The SCI rats immediately underwent open laparotomy and received detrusor injections of either BoNT-A (10 U/animal) or saline. At eight weeks post injury, the bladders were collected, weighed, and examined histologically. BoNT-A injected bladders of SCI rats (SCI + BoNT-A) weighed significantly less than saline injected bladders of SCI rats (SCI + saline) (241 ± 25 mg vs. 183 ± 42 mg; p < 0.05). Histological analyses showed that SCI resulted in significantly thicker bladder walls due to detrusor hypertrophy and fibrosis compared to bladders from uninjured animals (339 ± 89.0 µm vs. 193 ± 47.9 µm; p < 0.0001). SCI + BoNT-A animals had significantly thinner bladder walls compared to SCI + saline animals (202 ± 55.4 µm vs. 339 ± 89.0 µm; p < 0.0001). SCI + BoNT-A animals had collagen organization in the bladder walls similar to that of uninjured animals. Detrusor chemodenervation soon after SCI appears to preserve bladder tissue integrity by reducing the development of detrusor fibrosis and hypertrophy associated with SCI.

Viable human brain microvessels for the study of aging and neurodegenerative diseases.

The brain microvasculature is altered in normal aging and in the presence of disease processes, such as neurodegeneration or ischemia, but there are few methods for studying living tissues. We now report that viable microvessels (MV) are readily isolated from brain tissue of subjects enrolled in studies of neurodegenerative diseases who undergo rapid autopsy (performed with <12 h postmortem interval - PMI). We find that these MV retain their morphology and cellular components and are fairly uniform in size. Sufficient MV (~3-5000) are obtained from 3 to 4 g of tissue to allow for studies of cellular composition as well as extracellular matrix (ECM). Using live/dead assays, these MV are viable for up to 5 days in tissue culture media (2D) designed to support endothelial cells and up to 11 days post-isolation in a 3-dimensional (3D) matrix (Low Growth Factor Matrigel™). Assays that measure the reducing potential of live cells \demonstrated that the majority of the MV maintain high levels of metabolic activity for a similar number of days as the live/dead assays. Functional cellular components (such as tight junctions and transporter proteins) and ECM of MV in tissue culture media, and to a lesser extent in 3D matrices, were readily visualized using immunofluorescence techniques. MV in tissue culture media are lysed and protein content analyzed, but MV in 3D matrix first require removal of the supporting matrix, which can confound the analysis of MV ECM. Finally, MV can be preserved in cryoprotective media, whereby over 50% retain their baseline viability upon thawing. In summary, we find that MV isolated from human brains undergoing rapid autopsy are viable in standard tissue culture for up to 5 days and the timeframe for experiments can be extended up to 11 days by use of a supportive 3D matrix. Viable human MV allow for temporal and spatial analysis of relevant cellular and ECM components that have implications for microvascular function in neurodegenerative diseases, vascular brain injury, and neurotrauma.

Effect of Durotomy versus Myelotomy on Tissue Sparing and Functional Outcome after Spinal Cord Injury.

Various surgical strategies have been developed to alleviate elevated intraspinal pressure (ISP) following acute traumatic spinal cord injury (tSCI). Surgical decompression of either the dural (durotomy) or the dural and pial (myelotomy) lining of the spinal cord has been proposed. However, a direct comparison of these two strategies is lacking. Here, we compare the histological and functional effects of durotomy alone and durotomy plus myelotomy in a rodent model of acute thoracic tSCI. Our results indicate that tSCI causes local tissue edema and significantly elevates ISP (7.4 ± 0.3 mmHg) compared with physiological ISP (1.7 ± 0.4 mmHg; p < 0.001). Both durotomy alone and durotomy plus myelotomy effectively mitigate elevated local ISP (p < 0.001). Histological examination at 10 weeks after tSCI revealed that durotomy plus myelotomy promoted spinal tissue sparing by 13.7% compared with durotomy alone, and by 25.9% compared with tSCI-only (p < 0.0001). Both types of decompression surgeries elicited a significant beneficial impact on gray matter sparing (p < 0.01). Impressively, durotomy plus myelotomy surgery increased preservation of motor neurons by 174.3% compared with tSCI-only (p < 0.05). Durotomy plus myelotomy surgery also significantly promoted recovery of hindlimb locomotor function in an open-field test (p < 0.001). Interestingly, only durotomy alone resulted in favorable recovery of bladder and Ladder Walk performance. Combined, our data suggest that durotomy plus myelotomy following acute tSCI facilitates tissue sparing and recovery of locomotor function. In the future, biomarkers identifying spinal cord injuries that can benefit from either durotomy alone or durotomy plus myelotomy need to be developed.

Contrast-Enhanced Ultrasound for Assessment of Local Hemodynamic Changes Following a Rodent Contusion Spinal Cord Injury.

INTRODUCTION: Severe trauma to the spinal cord leads to a near complete loss of blood flow at the injury site along with significant hypoperfusion of adjacent tissues. Characterization and monitoring of local tissue hypoperfusion is currently not possible in clinical practice because available imaging techniques do not allow for assessment of blood flow with sufficient spatial and temporal resolutions. The objective of the current study was to determine whether ultrafast contrast-enhanced ultrasound (CEUS) imaging could be used to visualize and quantify acute hemodynamic changes in a rat traumatic spinal cord injury (SCI) model. MATERIALS AND METHODS: We used novel ultrasound acquisition and processing methods that allowed for measurements of local tissue perfusion as well as for assessment of structural and functional integrity of spinal vasculature. RESULTS: CEUS imaging showed that traumatic SCI results in (1) an area with significant loss of perfusion, which increased during the first hour after injury, (2) structural alterations of the spinal cord vasculature, and (3) significant slowing of arterial blood flow velocities around the injury epicenter. CONCLUSION: We conclude that CEUS has the spatial and temporal sensitivity and resolution to visualize local tissue perfusion and vessel architecture, which maybe useful clinically to determine injury extent and severity in patients with SCI.

Transcutaneous contrast-enhanced ultrasound imaging of the posttraumatic spinal cord.

STUDY DESIGN: Experimental animal study. OBJECTIVE: The current study aims to test whether the blood flow within the contused spinal cord can be assessed in a rodent model via the acoustic window of the laminectomy utilizing transcutaneous ultrasound. SETTING: Department of Neurological Surgery, University of Washington, Seattle WA. METHODS: Long-Evans rats (n = 12) were subjected to a traumatic thoracic spinal cord injury (SCI). Three days and 10 weeks after injury, animals underwent imaging of the contused spinal cord using ultrafast contrast-enhanced ultrasound with a Vantage ultrasound research system in combination with a 15 MHz transducer. Lesion size and signal-to-noise ratios were estimated via transcutaneous, subcutaneous, or epidural ultrasound acquisition through the acoustic window created by the original laminectomy. RESULTS: Following laminectomy, transcutaneous and subcutaneous contrast-enhanced ultrasound imaging allowed for assessment of perfusion and vascular flow in the contused rodent spinal cord. An average loss of 7.2 dB from transcutaneous to subcutaneous and the loss of 5.1 dB from subcutaneous to epidural imaging in signal-to-noise ratio (SNR) was observed. The hypoperfused injury center was measured transcutaneously, subcutaneously and epidurally (5.78 ± 0.86, 5.91 ± 0.53, 5.65 ± 1.07 mm2) at 3 days post injury. The same animals were reimaged again at 10 weeks following SCI, and the area of hypoperfusion had decreased significantly compared with the 3-day measurements detected via transcutaneous, subcutaneous, and epidural imaging respectively (0.69 ± 0.05, 1.09 ± 0.11, 0.95 ± 0.11 mm2, p < 0.001). CONCLUSIONS: Transcutaneous ultrasound allows for measurements and longitudinal monitoring of local hemodynamic changes in a rodent SCI model.

Accumbens brain-derived neurotrophic factor (BDNF) transmission inhibits cocaine seeking.

Brain-derived neurotrophic factor (BDNF) regulates a variety of physiological processes, and several studies have explored the role of BDNF in addiction-related brain regions like the nucleus accumbens core (NAcore). We sought to understand the rapid effects of endogenous BDNF on cocaine seeking. Rats were trained to self-administer cocaine and extinguished. We then microinjected two inhibitors of BDNF stimulation of tropomyosin receptor kinase B (TrkB), the non-competitive receptor antagonist ANA-12 and TrkB/Fc, a fusion protein that binds BDNF and prevents TrkB stimulation. Blocking TrkB or inactivating BDNF in NAcore potentiated active lever pressing, showing that endogenous BDNF tone was present and supplying inhibitory tone on cue-induced reinstatement. To determine if exogenous BDNF also negatively regulated reinstatement, BDNF was microinjected into NAcore 15 minutes before cue-induced reinstatement. BDNF decreased cocaine seeking through TrkB receptor binding, but had no effect on inactive lever pressing, spontaneous or cocaine-induced locomotion, or on reinstated sucrose seeking. BDNF-infusion potentiated within trial extinction when microinjected in the NAcore during cue- and context + cue induced reinstatement, and the inhibition of lever pressing lasted at least 3 days post injection. Although decreased reinstatement endured for 3 days when BDNF was administered prior to a reinstatement session, when microinjected before an extinction session or in the home cage, BDNF did not alter subsequent cued-reinstatement. Together, these data show that endogenous BDNF acts on TrKB to provide inhibitory tone on reinstated cocaine seeking, and this effect was recapitulated by exogenous BDNF.

Provisional crown failures in dental school predoctoral clinics.

Following a preliminary study indicating that at least 10 percent of single-unit crown temporary restorations failed in patients who received treatment by predoctoral students, a comprehensive examination of provisional crown failure was initiated to identify strategies to reduce the failure rate. For all provisionalized, natural tooth, single-unit crown preparations in University of North Carolina School of Dentistry predoctoral clinics for one year (N=1008), we noted tooth type, type of crown, student level, faculty coverage experience, treatment clinic, temporary material and luting agent, and retreatment (failure) of the provisional restoration. For failures, we also noted the stage of crown preparation at failure and the time since initial placement of the temporary. We analyzed these data using simple cross-tabs and logistic regression on need for retreatment (alpha =0.05). The failure rate was 18.75 percent (N=189). The median time to failure was twelve days; the 25(th) and 75(th) percentiles were six and twenty-six days. Significant risk factors, in order of odds ratio estimates, were molar tooth, second- or third-year student, and inexperienced faculty. Most provisional failures occurred during the final preparation phase of treatment. Provisional restoration failure is more frequent than was initially suspected from preliminary studies. Strategies for institutional intervention to reduce provisional restoration failure include greater attention to evaluating provisional crowns placed by inexperienced students (sophomores and juniors) and placing more emphasis on the retentiveness of provisional restorations reused following the final impression. Review of provisional evaluation procedures is also indicated for faculty who do not routinely supervise these procedures.