Investigation of Cellular and Molecular Responses to Pulsed Focused Ultrasound in a Mouse Model

Continuous focused ultrasound (cFUS) has been widely used for thermal ablation of tissues, relying on continuous exposures to generate temperatures necessary to induce coagulative necrosis. Pulsed FUS (pFUS) employs non-continuous exposures that lower the rate of energy deposition and allow cooling to occur between pulses, thereby minimizing thermal effects and emphasizing effects created by non-thermal mechanisms of FUS (i.e., acoustic radiation forces and acoustic cavitation). pFUS has shown promise for a variety of applications including drug and nanoparticle delivery; however, little is understood about the effects these exposures have on tissue, especially with regard to cellular pro-homing factors (growth factors, cytokines, and cell adhesion molecules). We examined changes in murine hamstring muscle following pFUS or cFUS and demonstrate that pFUS, unlike cFUS, has little effect on the histological integrity of muscle and does not induce cell death. Infiltration of macrophages was observed 3 and 8 days following pFUS or cFUS exposures. pFUS increased expression of several cytokines (e.g., IL-1α, IL-1β, TNFα, INFγ, MIP-1α, MCP-1, and GMCSF) creating a local cytokine gradient on days 0 and 1 post-pFUS that returns to baseline levels by day 3 post-pFUS. pFUS exposures induced upregulation of other signaling molecules (e.g., VEGF, FGF, PlGF, HGF, and SDF-1α) and cell adhesion molecules (e.g., ICAM-1 and VCAM-1) on muscle vasculature. The observed molecular changes in muscle following pFUS may be utilized to target cellular therapies by increasing homing to areas of pathology

Hypertension prevents a sensory stimulation-based collateral therapeutic from protecting the cortex from impending ischemic stroke damage in a spontaneously hypersensitive rat model.

Assessing potential stroke treatments in the presence of risk factors can improve screening of treatments prior to clinical trials and is important in testing the efficacy of treatments in different patient populations. Here, we test our noninvasive, nonpharmacological sensory stimulation treatment in the presence of the main risk factor for ischemic stroke, hypertension. Utilizing functional imaging, blood flow imaging, and histology, we assessed spontaneously hypertensive rats (SHRs) pre- and post-permanent middle cerebral artery occlusion (pMCAO). Experimental groups included a treatment SHR group (sensory-stimulated group), control untreated SHR group (no sensory stimulation), and a treated (sensory-stimulated) Wistar-Kyoto normotensive group. Unlike our previous studies, which showed sensory-based complete protection from impending ischemic cortical stroke damage in rats as seen in the treated Wistar-Kyoto group, we found that SHRs at 24hr post-pMCAO lacked evoked cortical activation, had a significant reduction in blood flow within the MCA, and sustained very large infarcts regardless of whether they received stimulation treatment. If translatable, this work highlights a potential need for a combined treatment plan when delivering sensory stimulation treatment in this patient population

Testing the effects of sensory stimulation as a collateral-based therapeutic for ischemic stroke in C57BL/6J and CD1 mouse strains.

Utilizing a rat model of ischemic stroke, we have previously shown that sensory stimulation can completely protect rats from impending ischemic damage of cortex if this treatment is delivered within the first two hours post-permanent middle cerebral artery occlusion (pMCAo). The current study sought to extend our findings in rats to mice, which would allow new avenues of research not available in rats. Thus, young adult C57BL/6J and CD1 mice were tested for protection from ischemic stroke with the same protective sensory stimulation-based treatment. Cortical activity and blood flow were assessed with intrinsic signal optical imaging (ISOI) and laser speckle imaging (LSI), respectively, and histological analysis (TTC) was performed at the completion of the experiments. Standing in stark contrast to the positive results observed in rats, in both strains we found that there were no differences between treated and untreated mice at 24 hours post-pMCAo in terms of infarct volume, negative functional imaging results, and major reduction in retrograde collateral blood flow as compared to pre-pMCAo baseline and surgical controls. Also, no differences were found between the strains in terms of theses variables. Potential reasons for the differences between rats and mice are discussed

Blood flow quantification for C57BL/6J and CD1 mice at baseline and 24 hours post-pMCAo as a percent of baseline flow.

<p>A, At baseline, C57BL/6J treated, untreated and surgical control groups were equivalent, but at 24 hours post-pMCAO, treated subjects were equivalent to untreated subjects and had significantly reduced blood flow within the MCA. Surgical controls maintained baseline levels of flow at 24 hours. B, Similar to C57BL/6J mice, CD1 treated and untreated subjects were equivalent and had significant reductions in blood flow at 24 hours, while surgical controls maintained baseline levels of flow at this time point. (** = p ≤ 0.01, *** = p ≤ 0.001).</p

WFR quantification for C57BL/6J and CD1 mice.

<p>A, WFRs were quantified in terms of their area and amplitude at baseline and 24 hours post-pMCAo. WFR quantification for C57BL/6J and CD1 mice shows that treated and untreated subjects had significant reductions in WFRs at 24 hours, while surgical controls maintained cortical activity. No differences were found between treated, untreated, and surgical control subjects across C57BL/6J and CD1 strains, indicating these groups were equivalent at baseline and 24 hours. (NS = not significant, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001). B, Two mice received full whisker array stimulation, and both subjects lacked WFR’s at 24 hours; TTC representative case shows infarct at 24 hours (black arrow).</p

TTC staining revealed no protective effect of treatment for either C57BL/6J or CD1 subjects.

<p>Infarct quantification for C57BL/6J and CD1 treated, untreated and surgical control groups. Despite receiving immediate treatment, treated subjects sustained infarct equivalent in size to untreated subjects. Surgical controls were significantly different from treated and untreated subjects as they did not sustain ischemic damage, but occasionally subjects would have evidence of surgical damage that was less than 1 mm³. Additionally, C57BL/6J and CD1 groups were equivalent for infarct volume. (NS = not significant, *** = p ≤ 0.001).</p

Treated C57BL/6J and CD1 mice are not protected from ischemic damage.

<p>A, Experimental schema with ISOI, LSI and TTC representative cases for C57BL/6J (green, blue, and yellow boxes) and CD1 mice (red, purple, and grey boxes): treated (top), untreated (middle), and surgical control (bottom) subjects, before (left) and 24 hours after (right) pMCAo. All subjects had whisker functional representations (WFRs) and blood flow within the MCA at baseline. When assessed at 24 hours post-pMCAo, treated and untreated subjects of both strains lacked WFRs, retrograde blood flow within the MCA was minimal, and TTC staining revealed infarct (right; see arrows). Surgical controls of both strains, however, maintained both WFRs and MCA blood flow, and did not sustain infarct since these subjects never received pMCAo. B, The imaging window (black-rimmed square) is centered over the barrel cortex (black regions), which is fed by major MCA branches (in red); the C2 whisker barrel is highlighted in red. The smaller representative images of LSI blood flow within the MCA are taken from a portion of a major MCA branch within this imaging window; the location in relation to C2 can vary between animals.</p

Sensory Stimulation-Based Complete Protection from Ischemic Stroke Remains Stable at 4 Months Post-Occlusion of MCA.

Previous research from our lab has shown that when using a rodent model of ischemic stroke (permanent middle cerebral artery occlusion), mild sensory stimulation, when delivered within two hours of ischemic onset, completely protects the cortex from impending ischemic stroke damage when assessed 24 hours post-occlusion. However, the long-term stability of this protection remains unclear. Using intrinsic signal optical imaging for assessment of cortical function, laser speckle imaging for assessment of blood flow, a battery of behavioral tests and cresyl violet for histological assessment, the present study examined whether this protection was long-lasting. When assessed 4 months post-occlusion (this length of time being equivalent to 10-15 years in humans), rats receiving sensory stimulation treatment immediately after ischemic onset exhibit normal neuronal and vascular function, and they are behaviorally and histologically equivalent to healthy controls (surgical shams). Thus, the complete neuroprotection due to cortical activation via sensory stimulation remains stable with time. These findings add support to the translational potential of this sensory stimulation-based treatment

Fully distributed absolute blood flow velocity measurement for middle cerebral arteries using Doppler optical coherence tomography.

Doppler optical coherence tomography (DOCT) is considered one of the most promising functional imaging modalities for neuro biology research and has demonstrated the ability to quantify cerebral blood flow velocity at a high accuracy. However, the measurement of total absolute blood flow velocity (BFV) of major cerebral arteries is still a difficult problem since it is related to vessel geometry. In this paper, we present a volumetric vessel reconstruction approach that is capable of measuring the absolute BFV distributed along the entire middle cerebral artery (MCA) within a large field-of-view. The Doppler angle at each point of the MCA, representing the vessel geometry, is derived analytically by localizing the artery from pure DOCT images through vessel segmentation and skeletonization. Our approach could achieve automatic quantification of the fully distributed absolute BFV across different vessel branches. Experiments on rodents using swept-source optical coherence tomography showed that our approach was able to reveal the consequences of permanent MCA occlusion with absolute BFV measurement