The mechanoelectrical transducer channel is not required for regulation of cochlear blood flow during loud sound exposure in mice.

The mammalian cochlea possesses unique acoustic sensitivity due to a mechanoelectrical 'amplifier', which requires the metabolic support of the cochlear lateral wall. Loud sound exposure sufficient to induce permanent hearing damage causes cochlear blood flow reduction, which may contribute to hearing loss. However, sensory epithelium involvement in the cochlear blood flow regulation pathway is not fully described. We hypothesize that genetic manipulation of the mechanoelectrical transducer complex will abolish sound induced cochlear blood flow regulation. We used salsa mice, a Chd23 mutant with no mechanoelectrical transduction, and deafness before p56. Using optical coherence tomography angiography, we measured the cochlear blood flow of salsa and wild-type mice in response to loud sound (120 dB SPL, 30 minutes low-pass filtered noise). An expected sound induced decrease in cochlear blood flow occurred in CBA/CaJ mice, but surprisingly the same sound protocol induced cochlear blood flow increases in salsa mice. Blood flow did not change in the contralateral ear. Disruption of the sympathetic nervous system partially abolished the observed wild-type blood flow decrease but not the salsa increase. Therefore sympathetic activation contributes to sound induced reduction of cochlear blood flow. Additionally a local, non-sensory pathway, potentially therapeutically targetable, must exist for cochlear blood flow regulation.

External Compression Versus Intravascular Injection: A Mechanistic Animal Model of Filler-Induced Tissue Ischemia.

PURPOSE: Soft tissue ischemia is a devastating and unpredictable complication following dermal filler injection. Multiple mechanisms to explain this complication have been proposed, including vascular compression, vessel damage, and intraarterial filler emboli. To elucidate the mechanism of injury, the authors introduce a mouse model, imaged with optical microangiography and laser speckle contrast imaging technologies, to demonstrate in vivo microvascular response to soft tissue and intravascular filler injection. METHODS: To determine the effect of external vascular compression on distal perfusion, the authors attempted to occlude vessels with subcutaneous hyaluronic acid gel (HAG) bolus injections into the pinna of hairless mice. The authors also performed suture ligation of a major vascular bundle. Following these interventions, laser speckle and optical microangiography were performed serially over 1 week follow up. To determine the effect of intravascular HAG injection, the authors devised and validated a novel method of cannulating the mouse external carotid artery for intraarterial access to the pinna vasculature. Using this model, the authors performed intraarterial HAG injections and completed optical microangiography and laser speckle contrast imaging. RESULTS: Despite large HAG bolus injections directly adjacent to vascular bundles, the authors were unable to induce compressive occlusion of the mouse pinna vessels. Vascular occlusion was successfully performed with suture ligation, but optical microangiography and laser speckle contrast imaging confirmed undisturbed distal capillary bed perfusion. With intravascular HAG injection, large segments of pinna showed distinct perfusion reduction along a vascular distribution when compared with preinjection images, most noticeably at the capillary level. CONCLUSIONS: The novel mouse pinna model combining intravascular access and in vivo microvascular perfusion imaging has furthered the understanding of the mechanism of filler-induced tissue ischemia. Distal capillary perfusion was maintained despite external vascular compression. Intraarterial HAG filler injection, however, resulted in large areas of capillary nonperfusion and is the most likely etiology for filler-induced tissue necrosis that is observed clinically.

Macro-to-micro cortical vascular imaging underlies regional differences in ischemic brain.

The ability to non-invasively monitor and quantify hemodynamic responses down to the capillary level is important for improved diagnosis, treatment and management of neurovascular disorders, including stroke. We developed an integrated multi-functional imaging system, in which synchronized dual wavelength laser speckle contrast imaging (DWLS) was used as a guiding tool for optical microangiography (OMAG) to test whether detailed vascular responses to experimental stroke in male mice can be evaluated with wide range sensitivity from arteries and veins down to the capillary level. DWLS enabled rapid identification of cerebral blood flow (CBF), prediction of infarct area and hemoglobin oxygenation over the whole mouse brain and was used to guide the OMAG system to hone in on depth information regarding blood volume, blood flow velocity and direction, vascular architecture, vessel diameter and capillary density pertaining to defined regions of CBF in response to ischemia. OMAG-DWLS is a novel imaging platform technology to simultaneously evaluate multiple vascular responses to ischemic injury, which can be useful in improving our understanding of vascular responses under pathologic and physiological conditions, and ultimately facilitating clinical diagnosis, monitoring and therapeutic interventions of neurovascular diseases.

Assessment of microcirculation dynamics during cutaneous wound healing phases in vivo using optical microangiography.

Cutaneous wound healing consists of multiple overlapping phases starting with blood coagulation following incision of blood vessels. We utilized label-free optical coherence tomography and optical microangiography (OMAG) to noninvasively monitor healing process and dynamics of microcirculation system in a mouse ear pinna wound model. Mouse ear pinna is composed of two layers of skin separated by a layer of cartilage and because its total thickness is around 500 µm, it can be utilized as an ideal model for optical imaging techniques. These skin layers are identical to human skin structure except for sweat ducts and glands. Microcirculatory system responds to the wound injury by recruiting collateral vessels to supply blood flow to hypoxic region. During the inflammatory phase, lymphatic vessels play an important role in the immune response of the tissue and clearing waste from interstitial fluid. In the final phase of wound healing, tissue maturation, and remodeling, the wound area is fully closed while blood vessels mature to support the tissue cells. We show that using OMAG technology allows noninvasive and label-free monitoring and imaging each phase of wound healing that can be used to replace invasive tissue sample histology and immunochemistry technologies.

Changes in cochlear blood flow in mice due to loud sound exposure measured with Doppler optical microangiography and laser Doppler flowmetry.

In this work we determined the contributions of loud sound exposure (LSE) on cochlear blood flow (CoBF) in an in vivo anesthetized mouse model. A broadband noise system (20 kHz bandwidth) with an intensity of 119 dB SPL, was used for a period of one hour to produce a loud sound stimulus. Two techniques were used to study the changes in blood flow, a Doppler optical microangiography (DOMAG) system; which can measure the blood flow within individual cochlear vessels, and a laser Doppler flowmetry (LDF) system; which averages the blood flow within a volume (a hemisphere of ~1.5 mm radius) of tissue. Both systems determined that the blood flow within the cochlea is reduced due to the LSE stimulation.

Tracking dynamic microvascular changes during healing after complete biopsy punch on the mouse pinna using optical microangiography.

Optical microangiography (OMAG) and Doppler optical microangiography (DOMAG) are two non-invasive techniques capable of determining the tissue microstructural content, microvasculature angiography, and blood flow velocity and direction. These techniques were used to visualize the acute and chronic microvascular and tissue responses upon an injury in vivo. A tissue wound was induced using a 0.5 mm biopsy punch on a mouse pinna. The changes in the microangiography, blood flow velocity and direction were quantified for the acute (<30 min) wound response and the changes in the tissue structure and microangiography were determined for the chronic wound response (30 min-60 days). The initial wound triggered recruitment of peripheral capillaries, as well as redirection of main arterial and venous blood flow within 3 min. The complex vascular networks and new vessel formation were quantified during the chronic response using fractal dimension. The highest rate of wound closure occurred between days 8 and 22. The vessel tortuosity increased during this time suggesting angiogenesis. Taken together, these data signify that OMAG has the capability to track acute and chronic changes in blood flow, microangiography and structure during wound healing. The use of OMAG has great potential to improve our understanding of vascular and tissue responses to injury in order to develop more effective therapeutics.

Monitoring hypoxia induced changes in cochlear blood flow and hemoglobin concentration using a combined dual-wavelength laser speckle contrast imaging and Doppler optical microangiography system.

A synchronized dual-wavelength laser speckle contrast imaging (DWLSCI) system and a Doppler optical microangiography (DOMAG) system was developed to determine several ischemic parameters in the cochlea due to a systemic hypoxic challenge. DWLSCI can obtain two-dimensional data, and was used to determine the relative changes in cochlear blood flow, and change in the concentrations of oxyhemoglobin (HbO), deoxyhemoglobin (Hb) and total hemoglobin (HbT) in mice. DOMAG can obtain three-dimensional data, and was used to determine the changes in cochlear blood flow with single vessel resolution. It was demonstrated that during a hypoxic challenge there was an increase in the concentrations of Hb, a decrease in the concentrations of HbO and cochlear blood flow, and a slight decrease in the concentration of HbT. Also, the rate of change in the concentrations of Hb and HbO was quantified during and after the hypoxic challenge. The ability to simultaneously measure these ischemic parameters with high spatio-temporal resolution will allow the detailed quantitative analysis of several hearing disorders, and will be useful for diagnosing and developing treatments.

Effects of hypoxia on cochlear blood flow in mice evaluated using Doppler optical microangiography.

Reduced cochlear blood flow (CoBF) is a main contributor to hearing loss. Studying CoBF has remained a challenge due to the lack of available tools. Doppler optical microangiography (DOMAG), a method to quantify single-vessel absolute blood flow, and laser Doppler flowmetry (LDF), a method for measuring the relative blood flow within a large volume of tissue, were used for determining the changes in CoBF due to systemic hypoxia in mice. DOMAG determined the change in blood flow in the apical turn (AT) with single-vessel resolution, while LDF averaged the change in the blood flow within a large volume of the cochlea (hemisphere with ∼1 to 1.5 mm radius). Hypoxia was induced by decreasing the concentration of oxygen-inspired gas, so that the oxygen saturation was reduced from >95% to ∼80%. DOMAG determined that during hypoxia the blood flow in two areas of the AT near and far from the helicotrema were increased and decreased, respectively. The LDF detected a decrease in blood flow within a larger volume of the cochlea (several turns averaged together). Therefore, the use of DOMAG as a tool for studying cochlear blood flow due to its ability to determine absolute flow values with single-vessel resolution was proposed.

Optical microangiography of retina and choroid and measurement of total retinal blood flow in mice.

We present a novel application of optical microangiography (OMAG) imaging technique for visualization of depth-resolved vascular network within retina and choroid as well as measurement of total retinal blood flow in mice. A fast speed spectral domain OCT imaging system at 820nm with a line scan rate of 140 kHz was developed to image the posterior segment of eyes in mice. By applying an OMAG algorithm to extract the moving blood flow signals out of the background tissue, we are able to provide true capillary level imaging of the retinal and choroidal vasculature. The microvascular patterns within different retinal layers are presented. An en face Doppler OCT approach [Srinivasan et al., Opt Express 18, 2477 (2010)] was adopted for retinal blood flow measurement. The flow is calculated by integrating the axial blood flow velocity over the vessel area measured in an en face plane without knowing the blood vessel angle. Total retinal blood flow can be measured from both retinal arteries and veins. The results indicate that OMAG has the potential for qualitative and quantitative evaluation of the microcirculation in posterior eye compartments in mouse models of retinopathy and neovascularization.

Fast synchronized dual-wavelength laser speckle imaging system for monitoring hemodynamic changes in a stroke mouse model.

In this Letter, we describe a newly developed synchronized dual-wavelength laser speckle contrast imaging system, which contains two cameras that are synchronously triggered to acquire data. The system can acquire data at a high spatiotemporal resolution (up to 500 Hz for ~1000×1000 pixels). A mouse model of stroke is used to demonstrate the capability for imaging the fast changes (within tens of milliseconds) in oxygenated and deoxygenated hemoglobin concentration, and the relative changes in blood flow in the mouse brain, through an intact cranium. This novel imaging technology will enable the study of fast hemodynamics and metabolic changes in vascular diseases.

Quantifying optical microangiography images obtained from a spectral domain optical coherence tomography system.

The blood vessel morphology is known to correlate with several diseases, such as cancer, and is important for describing several tissue physiological processes, like angiogenesis. Therefore, a quantitative method for characterizing the angiography obtained from medical images would have several clinical applications. Optical microangiography (OMAG) is a method for obtaining three-dimensional images of blood vessels within a volume of tissue. In this study we propose to quantify OMAG images obtained with a spectral domain optical coherence tomography system. A technique for determining three measureable parameters (the fractal dimension, the vessel length fraction, and the vessel area density) is proposed and validated. Finally, the repeatability for acquiring OMAG images is determined, and a new method for analyzing small areas from these images is proposed.

Hemodynamic and morphological vasculature response to a burn monitored using a combined dual-wavelength laser speckle and optical microangiography imaging system.

A multi-functional imaging system capable of determining relative changes in blood flow, hemoglobin concentration, and morphological features of the blood vasculature is demonstrated. The system combines two non-invasive imaging techniques, a dual-wavelength laser speckle contrast imaging (2-LSI) and an optical microangiography (OMAG) system. 2-LSI is used to monitor the changes in the dynamic blood flow and the changes in the concentration of oxygenated (HbO), deoxygenated (Hb) and total hemoglobin (HbT). The OMAG system is used to acquire high resolution images of the functional blood vessel network. The vessel area density (VAD) is used to quantify the blood vessel network morphology, specifically the capillary recruitment. The proposed multi-functional system is employed to assess the blood perfusion status from a mouse pinna before and immediately after a burn injury. To our knowledge, this is the first non-invasive, non-contact and multifunctional imaging modality that can simultaneously measure variations of several blood perfusion parameters.

Responses of peripheral blood flow to acute hypoxia and hyperoxia as measured by optical microangiography.

Oxygen availability is regarded as a critical factor to metabolically regulate systemic blood flow. There is a debate as to how peripheral blood flow (PBF) is affected and modulated during hypoxia and hyperoxia; however in vivo evaluating of functional PBF under oxygen-related physiological perturbation remains challenging. Microscopic observation, the current frequently used imaging modality for PBF characterization often involves the use of exogenous contrast agents, which would inevitably perturb the intrinsic physiologic responses of microcirculation being investigated. In this paper, optical micro-angiography (OMAG) was employed that uses intrinsic optical scattering signals backscattered from blood flows for imaging PBF in skeletal muscle challenged by the alteration of oxygen concentration. By utilizing optical reflectance signals, we demonstrated that OMAG is able to show the response of hemodynamic activities upon acute hypoxia and hyperoxia, including the modulation of macrovascular caliber, microvascular density, and flux regulation within different sized vessels within skeletal muscle in mice in vivo. Our results suggest that OMAG is a promising tool for in vivo monitoring of functional macro- or micro-vascular responses within peripheral vascular beds.

Recombinant T cell receptor ligands improve outcome after experimental cerebral ischemia.

A key target for novel stroke therapy is the regulation of post-ischemic inflammatory mechanisms. Recent evidence emphasizes the role of T lymphocytes of differing subtypes in the evolution is ischemic brain damage. We have recently demonstrated the benefit of myelin antigen-specific immunodulatory agents known as recombinant T cell receptor ligands (RTLs) in a standard murine model of focal stroke. The aim of the current study was to extend this initial observation to RTL treatment in a therapeutically relevant timing after middle cerebral artery occlusion (MCAO) and verify functional benefit to complement histological outcome measures. We observed that the administration of mouse-specific RTL551 reduced infarct size and improved sensorimotor outcome when administered within a 3 h post-ischemic therapeutic window. RTL551 treatment reduced cortical, caudate putamen, and total infarct volume as compared to vehicle-treated mice. Using a standard behavioral testing repertoire, we observed that RTL551 reduced sensorimotor impairment 3 days after MCAO. Humanized RTL1000 (HLA-DR2 moiety linked to hMOG-35-55 peptide) also reduced infarct size in HLA-DR2 transgenic mice. These data indicate that this neuroantigen-specific immunomodulatory agent reduces damage when administered in a therapeutically relevant reperfusion timeframe.

Regulatory B cells limit CNS inflammation and neurologic deficits in murine experimental stroke.

Evaluation of infarct volumes and infiltrating immune cell populations in mice after middle cerebral artery occlusion (MCAO) strongly implicates a mixture of both pathogenic and regulatory immune cell subsets in stroke pathogenesis and recovery. Our goal was to evaluate the contribution of B cells to the development of MCAO by comparing infarct volumes and functional outcomes in wild-type (WT) versus B-cell-deficient µMT(-/-) mice. The results clearly demonstrate larger infarct volumes, higher mortality, more severe functional deficits, and increased numbers of activated T cells, macrophages, microglial cells, and neutrophils in the affected brain hemisphere of MCAO-treated µMT(-/-) versus WT mice. These MCAO-induced changes were completely prevented in B-cell-restored µMT(-/-) mice after transfer of highly purified WT GFP(+) B cells that were detected in the periphery, but not the CNS. In contrast, transfer of B cells from IL-10(-/-) mice had no effect on infarct volume when transferred into µMT(-/-) mice. These findings strongly support a previously unrecognized activity of IL-10-secreting WT B cells to limit infarct volume, mortality rate, recruitment of inflammatory cells, and functional neurological deficits 48 h after MCAO. Our novel observations are the first to implicate IL-10-secreting B cells as a major regulatory cell type in stroke and suggest that enhancement of regulatory B cells might have application as a novel therapy for this devastating neurologic condition.

Therapy with recombinant T-cell receptor ligand reduces infarct size and infiltrating inflammatory cells in brain after middle cerebral artery occlusion in mice.

Stroke induces a biphasic effect on the peripheral immune response that involves early activation of peripheral leukocytes followed by severe immunosuppression and atrophy of the spleen. Peripheral immune cells, including T lymphocytes, migrate to the brain and exacerbate the developing infarct. Recombinant T-cell receptor (TCR) Ligand (RTL)551 is designed as a partial TCR agonist for myelin oligodendrocyte glycoprotein (MOG)-reactive T cells and has demonstrated the capacity to limit infarct volume and inflammation in brain when administered to mice undergoing middle cerebral artery occlusion (MCAO). The goal of this study was to determine if RTL551 could retain protection when given within the therapeutically relevant 4 h time window currently in clinical practice for stroke patients. RTL551 was administered subcutaneously 4 h after MCAO, with repeated doses every 24 h until the time of euthanasia. Cell numbers were assessed in the brain, blood, spleen and lymph nodes and infarct size was measured after 24 and 96 h reperfusion. RTL551 reduced infarct size in both cortex and striatum at 24 h and in cortex at 96 h after MCAO and inhibited the accumulation of inflammatory cells in brain at both time points. At 24 h post-MCAO, RTL551 reduced the frequency of the activation marker, CD44, on T-cells in blood and in the ischemic hemisphere. Moreover, RTL551 reduced expression of the chemokine receptors, CCR5 in lymph nodes and spleen, and CCR7 in the blood and lymph nodes. These data demonstrate effective treatment of experimental stroke with RTL551 within a therapeutically relevant 4 h time window through immune regulation of myelin-reactive inflammatory T-cells.

Role of dihydrotestosterone in post-stroke peripheral immunosuppression after cerebral ischemia.

Stroke is a sexually dimorphic disease with male gender considered a disadvantage in terms of risk and disease outcome. In intact males, stroke induces peripheral immunosuppression, characterized by decreased splenocyte numbers and proliferation and altered percentages of viable T, B, and CD11b+ cells. To investigate whether the potent androgen and known immunomodulator, dihydrotestosterone (DHT), exacerbates post-stroke immunosuppression in castrated male mice after focal stroke, we evaluated the effect of middle cerebral artery occlusion (MCAO) on peripheral and central nervous system (CNS) immune responses in castrated mice with or without controlled levels of DHT. MCAO reduced spleen cell numbers in both groups, but altered T cell and B cell percentages in remaining splenocytes and concomitantly increased the percentage of CD11b+ blood cells solely in DHT-replaced animals at 24 h. Furthermore, DHT-replacement reduced splenocyte proliferation which was accompanied by an increased percentage of immunosuppressive regulatory T cells relative to castrates 96 h post-MCAO. In brain, the percentages of immune cell populations in the ischemic hemisphere relative to the non-ischemic hemisphere were similar between castrated and DHT-replaced mice after MCAO. These data suggest DHT modulates peripheral immunosuppression after MCAO but with relatively little effect on early immune response of the recovering CNS.

Involvement of Stat3 in mouse brain development and sexual dimorphism: a proteomics approach.

Although the role of STAT3 in cell physiology and tissue development has been largely investigated, its involvement in the development and maintenance of nervous tissue and in the mechanisms of neuroprotection is not yet known. The potentially wide range of STAT3 activities raises the question of tissue- and gender-specificity as putative mechanisms of regulation. To explore the function of STAT3 in the brain and the hypothesis of a gender-linked modulation of STAT3, we analyzed a neuron-specific STAT3 knockout mouse model investigating the influence of STAT3 activity in brain protein expression pattern in both males and females in the absence of neurological insult. We performed a proteomic study aimed to reveal the molecular pathways directly or indirectly controlled by STAT3 underscoring its role in brain development and maintenance. We identified several proteins, belonging to different neuronal pathways such as energy metabolism or synaptic transmission, controlled by STAT3 that confirm its crucial role in brain development and maintenance. Moreover, we investigated the different processes that could contribute to the sexual dimorphic behavior observed in the incidence of neurological and mental disease. Interestingly both STAT3 KO and gender factors influence the expression of several mitochondrial proteins conferring to mitochondrial activity high importance in the regulation of brain physiology and conceivable relevance as therapeutic target.

Estradiol and G1 reduce infarct size and improve immunosuppression after experimental stroke.

Reduced risk and severity of stroke in adult females is thought to depend on normal endogenous levels of estrogen, a well-known neuroprotectant and immunomodulator. In male mice, experimental stroke induces immunosuppression of the peripheral immune system, characterized by a reduction in spleen size and cell numbers and decreased cytokine and chemokine expression. However, stroke-induced immunosuppression has not been evaluated in female mice. To test the hypothesis that estradiol (E2) deficiency exacerbates immunosuppression after focal stroke in females, we evaluated the effect of middle cerebral artery occlusion on infarct size and peripheral and CNS immune responses in ovariectomized mice with or without sustained, controlled levels of 17-beta-E2 administered by s.c. implant or the putative membrane estrogen receptor agonist, G1. Both E2- and G1-replacement decreased infarct volume and partially restored splenocyte numbers. Moreover, E2-replacement increased splenocyte proliferation in response to stimulation with anti-CD3/CD28 Abs and normalized aberrant mRNA expression for cytokines, chemokines, and chemokine receptors and percentage of CD4(+)CD25(+)FoxP3(+) T regulatory cells observed in E2-deficient animals. These beneficial changes in peripheral immunity after E2 replacement were accompanied by a profound reduction in expression of the chemokine, MIP-2, and a 40-fold increased expression of CCR7 in the lesioned brain hemisphere. These results demonstrate for the first time that E2 replacement in ovariectomized female mice improves stroke-induced peripheral immunosuppression.

Mechanisms of gender-linked ischemic brain injury.

Biological sex is an important determinant of stroke risk and outcome. Women are protected from cerebrovascular disease relative to men, an observation commonly attributed to the protective effect of female sex hormones, estrogen and progesterone. However, sex differences in brain injury persist well beyond the menopause and can be found in the pediatric population, suggesting that the effects of reproductive steroids may not completely explain sexual dimorphism in stroke. We review recent advances in our understanding of sex steroids (estradiol, progesterone and testosterone) in the context of ischemic cell death and neuroprotection. Understanding the molecular and cell-based mechanisms underlying sex differences in ischemic brain injury will lead to a better understanding of basic mechanisms of brain cell death and is an important step toward designing more effective therapeutic interventions in stroke.