Immune cell identity behind the Ktrans mapping of mouse glioblastoma.

Dynamic contrast-enhanced MR imaging (DCE-MRI) can assess the integrity of the blood brain barrier (BBB) and has been used in GBM patients to determine glioma grade, predict prognosis, evaluate treatment response, and differentiate treatment-induced effect from recurrence. The volume transfer constant Ktrans is the most frequently used metric in tumor assessment. Based on previous studies that a higher WHO grade of brain tumor was associated with greater impairments of immunity and that Ktrans value was associated with the pathological grading, the relationship between differential composition of immune cells in GBM tissue and dynamic changes in Ktrans mapping was anticipated in this study. The present study utilized an orthotopic allograft model of GBM in which mouse GL26 cells are implanted into Ccr2RFP/wtCx3cr1GFP/wt mice on a C57 background. The brain tumors exhibited heterogenous Ktrans values with the coefficients of variation (CV) above 75%, or relatively homogeneous Ktrans maps with CV values below 50%. The Ktrans values of homogeneous tumors ranged between 0.02/min-0.32/min with a median value of 0.10/min. The immune cell composition defined by quantitative immunohistochemistry and cell sorting was compared between the tumors with Ktrans values above 0.10/min (higher Ktrans) or below 0.10/min (lower Ktrans). Histological analysis showed that tumors with higher Ktrans values exhibited greater numbers of CCR2pos cells (257.60 ± 16.42/mm2 vs 203.23 ± 12.20/mm2, p = 0.04) and an increased ratio of CCR2pos cells to CX3CR1pos cells (1.20 ± 0.02 vs 0.38 ± 0.04, p = 0.001), the numbers of CX3CR1pos cells did not differ significantly based on Ktrans values (219.70 ± 16.20/mm2 vs 250.38 ± 21.20/mm2, p = 0.19). Flowcytometry analysis showed that tumors with higher Ktrans values (above 0.1/min) were associated with greater numbers of both overall monocytes (54.93 ± 6.81% vs 29.75 ± 3.54%, p = 0.01) and inflammatory monocytes (72.38 ± 1.49% vs 59.52 ± 2.44%, p = 0.001). In contrast, tumors with lower Ktrans values (below 0.1/min) exhibited greater numbers of patrolling monocytes (75.65 ± 4.14% vs 63 ± 6.94%, p = 0.05). In the tumors with lower Ktrans values, all three types of tumor associated cells, including patrolling monocytes, inflammatory monocytes, and microglia cells possessed a higher proportion of cells at pro-inflammatory status (41.77 ± 6.13% vs 25.06 ± 6.72%, p = 0.05; 27.50 ± 2.11% vs 20.62 ± 1.87%, p = 0.03; and 55.80 ± 9.88% vs 31.12 ± 7.31%, p = 0.05), inflammatory monocytes showed fewer anti-inflammatory cells (1.25 ± 0.62% vs 3.16 ± 3.56%, p = 0.04). Taken together, differences in Ktrans values were associated with differential immune cell phenotypes and polarizations. Ktrans mapping may therefore represent a novel approach for defining the immune status of GBM.

Improving in situ acoustic intensity estimates using MR acoustic radiation force imaging in combination with multifrequency MR elastography.

PURPOSE: Magnetic resonance acoustic radiation force imaging (MR-ARFI) enables focal spot localization during nonablative transcranial ultrasound therapies. As the acoustic radiation force is proportional to the applied acoustic intensity, measured MR-ARFI displacements could potentially be used to estimate the acoustic intensity at the target. However, variable brain stiffness is an obstacle. The goal of this study was to develop and assess a method to accurately estimate the acoustic intensity at the focus using MR-ARFI displacements in combination with viscoelastic properties obtained with multifrequency MR elastography (MRE). METHODS: Phantoms with a range of viscoelastic properties were fabricated, and MR-ARFI displacements were acquired within each phantom using multiple acoustic intensities. Voigt model parameters were estimated for each phantom based on storage and loss moduli measured using multifrequency MRE, and these were used to predict the relationship between acoustic intensity and measured displacement. RESULTS: Using assumed viscoelastic properties, MR-ARFI displacements alone could not accurately estimate acoustic intensity across phantoms. For example, acoustic intensities were underestimated in phantoms stiffer than the assumed stiffness and overestimated in phantoms softer than the assumed stiffness. This error was greatly reduced using individualized viscoelasticity measurements obtained from MRE. CONCLUSION: We demonstrated that viscoelasticity information from MRE could be used in combination with MR-ARFI displacements to obtain more accurate estimates of acoustic intensity. Additionally, Voigt model viscosity parameters were found to be predictive of the relaxation rate of each phantom's time-varying displacement response, which could be used to optimize patient-specific MR-ARFI pulse sequences.

Non-invasive, neurotoxic surgery reduces seizures in a rat model of temporal lobe epilepsy.

Surgery can be highly effective for treating certain cases of drug resistant epilepsy. The current study tested a novel, non-invasive, surgical strategy for treating seizures in a rat model of temporal lobe epilepsy. The surgical approach uses magnetic resonance-guided, low-intensity focused ultrasound (MRgFUS) in combination with intravenous microbubbles to open the blood-brain barrier (BBB) in a transient and focal manner. During the period of BBB opening, a systemically administered neurotoxin (Quinolinic Acid: QA) that is normally impermeable to the BBB gains access to a targeted area in the brain, destroying neurons where the BBB has been opened. This strategy is termed Precise Intracerebral Non-invasive Guided Surgery (PING). Spontaneous recurrent seizures induced by pilocarpine were monitored behaviorally prior to and after PING or under control conditions. Seizure frequency in untreated animals or animals treated with MRgFUS without QA exhibited expected seizure rate fluctuations frequencies between the monitoring periods. In contrast, animals treated with PING targeting the intermediate-temporal aspect of the hippocampus exhibited substantial reductions in seizure frequency, with convulsive seizures being eliminated entirely in two animals. These findings suggest that PING could provide a useful alternative to invasive surgical interventions for treating drug resistant epilepsy, and perhaps for treating other neurological disorders in which aberrant neural circuitries play a role.

Histologic evaluation of activation of acute inflammatory response in a mouse model following ultrasound-mediated blood-brain barrier using different acoustic pressures and microbubble doses.

Rationale: Localized blood-brain barrier (BBB) opening can be achieved with minimal to no tissue damage by applying pulsed focused ultrasound alongside a low microbubble (MB) dose. However, relatively little is known regarding how varying treatment parameters affect the degree of neuroinflammation following BBB opening. The goal of this study was to evaluate the activation of an inflammatory response following BBB opening as a function of applied acoustic pressure using two different microbubble doses. Methods: Mice were treated with 650 kHz ultrasound using varying acoustic peak negative pressures (PNPs) using two different MB doses, and activation of an inflammatory response, in terms of microglial and astrocyte activation, was assessed one hour following BBB opening using immunohistochemical staining. Harmonic and subharmonic acoustic emissions (AEs) were monitored for all treatments with a passive cavitation detector, and contrast-enhanced magnetic resonance imaging (CE-MRI) was performed following BBB opening to quantify the degree of opening. Hematoxylin and eosin-stained slides were assessed for the presence of microhemorrhage and edema. Results: For each MB dose, BBB opening was achieved with minimal activation of microglia and astrocytes using a PNP of 0.15 MPa. Higher PNPs were associated with increased activation, with greater increases associated with the use of the higher MB dose. Additionally, glial activation was still observed in the absence of histopathological findings. We found that CE-MRI was most strongly correlated with the degree of activation. While acoustic emissions were not predictive of microglial or astrocyte activation, subharmonic AEs were strongly associated with marked and severe histopathological findings. Conclusions: Our study demonstrated that there were mild histologic changes and activation of the acute inflammatory response using PNPs ranging from 0.15 MPa to 0.20 MPa, independent of MB dose. However, when higher PNPs of 0.25 MPa or above were applied, the same applied PNP resulted in more severe and widespread histological findings and activation of the acute inflammatory response when using the higher MB dose. The potential activation of the inflammatory response following ultrasound-mediated BBB opening should be considered when treating patients to maximize therapeutic benefit.

Histologic safety of transcranial focused ultrasound neuromodulation and magnetic resonance acoustic radiation force imaging in rhesus macaques and sheep.

BACKGROUND: Neuromodulation by transcranial focused ultrasound (FUS) offers the potential to non-invasively treat specific brain regions, with treatment location verified by magnetic resonance acoustic radiation force imaging (MR-ARFI). OBJECTIVE: To investigate the safety of these methods prior to widespread clinical use, we report histologic findings in two large animal models following FUS neuromodulation and MR-ARFI. METHODS: Two rhesus macaques and thirteen Dorset sheep were studied. FUS neuromodulation was targeted to the primary visual cortex in rhesus macaques and to subcortical locations, verified by MR-ARFI, in eleven sheep. Both rhesus macaques and five sheep received a single FUS session, whereas six sheep received repeated sessions three to six days apart. The remaining two control sheep did not receive ultrasound but otherwise underwent the same anesthetic and MRI procedures as the eleven experimental sheep. Hematoxylin and eosin-stained sections of brain tissue (harvested zero to eleven days following FUS) were evaluated for tissue damage at FUS and control locations as well as tissue within the path of the FUS beam. TUNEL staining was used to evaluate for the presence of apoptosis in sheep receiving high dose FUS. RESULTS: No FUS-related pre-mortem histologic findings were observed in the rhesus macaques or in any of the examined sheep. Extravascular red blood cells (RBCs) were present within the meninges of all sheep, regardless of treatment group. Similarly, small aggregates of perivascular RBCs were rarely noted in non-target regions of neural parenchyma of FUS-treated (8/11) and untreated (2/2) sheep. However, no concurrent histologic abnormalities were observed, consistent with RBC extravasation occurring as post-mortem artifact following brain extraction. Sheep within the high dose FUS group were TUNEL-negative at the targeted site of FUS. CONCLUSIONS: The absence of FUS-related histologic findings suggests that the neuromodulation and MR-ARFI protocols evaluated do not cause tissue damage.

Effects of Non-invasive, Targeted, Neuronal Lesions on Seizures in a Mouse Model of Temporal Lobe Epilepsy.

Surgery to treat drug-resistant epilepsy can be quite effective but remains substantially underutilized. A pilot study was undertaken to test the feasibility of using a non-invasive, non-ablative, approach to produce focal neuronal loss to treat seizures in a rodent model of temporal lobe epilepsy. In this study, spontaneous, recurrent seizures were established in a mouse model of pilocarpine-induced status epilepticus. After post-status epilepticus stabilization, baseline behavioral seizures were monitored for 30 d. Non-invasive opening of the blood-brain barrier targeting the hippocampus was then produced by using magnetic resonance-guided, low-intensity focused ultrasound, through which a neurotoxin (quinolinic acid) administered intraperitoneally gained access to the brain parenchyma to produce focal neuronal loss. Behavioral seizures were then monitored for 30 d after this procedure, and brains were subsequently prepared for histologic analysis of the sites of neuronal loss. The average frequency of behavioral seizures in all animals (n = 11) was reduced by 21.2%. Histologic analyses along the longitudinal axis of the hippocampus revealed that most of the animals (n = 8) exhibited neuronal loss located primarily in the intermediate aspect of the hippocampus, while sparing the septal aspect. Two other animals with damage to the intermediate hippocampus also exhibited prominent bilateral damage to the septal aspect of the hippocampus. A final animal had negligible neuronal loss overall. Notably, the site of neuronal loss along the longitudinal axis of the hippocampus influenced seizure outcomes. Animals that did not have bilateral damage to the septal hippocampus displayed a mean decrease in seizure frequency of 27.7%, while those with bilateral damage to the septal hippocampus actually increased seizure frequency by 18.7%. The animal without neuronal loss exhibited an increase in seizure frequency of 19.6%. The findings indicate an overall decrease in seizure frequency in treated animals. And, the site of neuronal loss along the longitudinal axis of the hippocampus appears to play a key role in reducing seizure activity. These pilot data are promising, and they encourage additional and more comprehensive studies examining the effects of targeted, non-invasive, neuronal lesions for the treatment of epilepsy.