Cardiomyocyte Alpha-1A Adrenergic Receptors Mitigate Postinfarct Remodeling and Mortality by Constraining Necroptosis.

Clinical studies have shown that α1-adrenergic receptor antagonists (α-blockers) are associated with increased heart failure risk. The mechanism underlying that hazard and whether it arises from direct inhibition of cardiomyocyte α1-ARs or from systemic effects remain unclear. To address these issues, we created a mouse with cardiomyocyte-specific deletion of the α1A-AR subtype and found that it experienced 70% mortality within 7 days of myocardial infarction driven, in part, by excessive activation of necroptosis. We also found that patients taking α-blockers at our center were at increased risk of death after myocardial infarction, providing clinical correlation for our translational animal models.

Mouse Brain MRI: Including In Vivo, Ex Vivo, and fcMRI for the Study of Microcephaly.

With its sensitivity to soft tissue, MRI is a powerful tool for the study of the neuroanatomical manifestations of a variety of conditions, such as microcephaly-related morbidities that are not easily visualized by other imaging techniques, such as CT. In addition to structural imaging, more recently, researchers have found changes in brain function in a wide range of neurological conditions-highlighting the utility of MRI for the study of microcephaly.In this methods chapter, basic mouse preparation and the acquisition of data for in vivo anatomical MRI will be discussed. Additionally, we will provide our protocol for the perfusion and fixation of brain tissue with gadolinium contrast agent. Following that, the process of optimization of system parameters will be shown for anatomical imaging of in vivo and ex vivo brain tissue. Lastly, the chapter will detail a protocol for fcMRI along with a discussion of considerations specific to functional imaging.

Computing hemodynamic response functions from concurrent spectral fiber-photometry and fMRI data.

Significance: Although emerging evidence suggests that the hemodynamic response function (HRF) can vary by brain region and species, a single, canonical, human-based HRF is widely used in animal studies. Therefore, the development of flexible, accessible, brain-region specific HRF calculation approaches is paramount as hemodynamic animal studies become increasingly popular. Aim: To establish an fMRI-compatible, spectral, fiber-photometry platform for HRF calculation and validation in any rat brain region. Approach: We used our platform to simultaneously measure (a) neuronal activity via genetically encoded calcium indicators (GCaMP6f), (b) local cerebral blood volume (CBV) from intravenous Rhodamine B dye, and (c) whole brain CBV via fMRI with the Feraheme contrast agent. Empirical HRFs were calculated with GCaMP6f and Rhodamine B recordings from rat brain regions during resting-state and task-based paradigms. Results: We calculated empirical HRFs for the rat primary somatosensory, anterior cingulate, prelimbic, retrosplenial, and anterior insular cortical areas. Each HRF was faster and narrower than the canonical HRF and no significant difference was observed between these cortical regions. When used in general linear model analyses of corresponding fMRI data, the empirical HRFs showed better detection performance than the canonical HRF. Conclusions: Our findings demonstrate the viability and utility of fiber-photometry-based HRF calculations. This platform is readily scalable to multiple simultaneous recording sites, and adaptable to study transfer functions between stimulation events, neuronal activity, neurotransmitter release, and hemodynamic responses.

IL-11 antagonist suppresses Th17 cell-mediated neuroinflammation and demyelination in a mouse model of relapsing-remitting multiple sclerosis.

IL-11 induced differentiation and expansion of Th17 cells in patients with early relapsing-remitting multiple sclerosis (RRMS). In mice with relapsing-remitting experimental autoimmune encephalomyelitis (RREAE), IL-11 exacerbated disease, induced demyelination in the central nervous system (CNS), increased the percentage of IL-17A+CD4+ Th17 cells in the CNS in the early acute phase, and up-regulated serum IL-17A levels and the percentage of IL-17A+CD4+ Th17 cells in lymph nodes, and IFN-γ+CD4+ T cells in spinal cord in the RR phase. IL-11 antagonist suppressed RREAE disease activities, inhibited IL-17A+CD4+ cell infiltration and demyelination in the CNS, and decreased the percentage of IL-17A+CD4+ T cells in peripheral blood mononuclear cells and ICAM1+CD4+ T cells in brain and SC. Diffusion Tensor Imaging indicated that IL-11 antagonist inhibited demyelination in several brain regions. We conclude that by suppressing Th17 cell-mediated neuroinflammation and demyelination, IL-11 antagonist can be further studied as a potential selective and early therapy for RRMS.

Structural and functional connectivity between the lateral posterior-pulvinar complex and primary visual cortex in the ferret.

The role of higher-order thalamic structures in sensory processing remains poorly understood. Here, we used the ferret (Mustela putorius furo) as a novel model species for the study of the lateral posterior (LP)-pulvinar complex and its structural and functional connectivity with area 17 [primary visual cortex (V1)]. We found reciprocal anatomical connections between the lateral part of the LP nucleus of the LP-pulvinar complex (LPl) and V1. In order to investigate the role of this feedback loop between LPl and V1 in shaping network activity, we determined the functional interactions between LPl and the supragranular, granular and infragranular layers of V1 by recording multiunit activity and local field potentials. Coherence was strongest between LPl and the supragranular V1, with the most distinct peaks in the delta and alpha frequency bands. Inter-area interaction measured by spike-phase coupling identified the delta frequency band being dominated by the infragranular V1 and multiple frequency bands that were most pronounced in the supragranular V1. This inter-area coupling was differentially modulated by full-field synthetic and naturalistic visual stimulation. We also found that visual responses in LPl were distinct from those in V1 in terms of their reliability. Together, our data support a model of multiple communication channels between LPl and the layers of V1 that are enabled by oscillations in different frequency bands. This demonstration of anatomical and functional connectivity between LPl and V1 in ferrets provides a roadmap for studying the interaction dynamics during behaviour, and a template for identifying the activity dynamics of other thalamo-cortical feedback loops.

Comparison of retinal and cerebral blood flow between continuous arterial spin labeling MRI and fluorescent microsphere techniques.

PURPOSE: To compare basal retinal and cerebral blood flow (BF) values using continuous arterial spin labeling (CASL) MRI and fluorescent microspheres. MATERIALS AND METHODS: A total of 41 animals were used. BF was measured using an established microsphere technique (a mixture of 2.5 million 8 µm green and 0.5 million 10 µm blue fluorescent microspheres) and CASL MRI blood flow measurement in the rat retina and brain at 7 Tesla (T) and 11.7T, respectively. RESULTS: Retinal BF by MRI was 1.18 ± 0.57 mL/g/min and choroidal BF was 8.14 ± 1.8 mL/g/min (n = 6). Microsphere retinal BF was 9.12 ± 2.8 µL/min per tissue and choroidal BF was 73.38 ± 44 µL/min per tissue (n = 18), corresponding to a retinal BF value of 1.22 ± 0.36 mL/g/min by means of a wet weight conversion. The wet-weight of the choroid could not be determined. To corroborate our findings, cerebral BF (CBF) by MRI was also analyzed. In the cerebral cortices, CBF was 0.91 ± 0.29 mL/g/min (n = 14) by CASL MRI and 1.09 ± 0.37 mL/g/min (n = 6) by microspheres. There were no significant differences found between MRI and microsphere blood flow in the retina and brain. CONCLUSION: BF values in the rat retina and cerebral cortex by MRI are in agreement with those obtained by the microsphere technique.

Methylene blue potentiates stimulus-evoked fMRI responses and cerebral oxygen consumption during normoxia and hypoxia.

Methylene blue USP (MB) at low doses has metabolic-enhancing and antioxidant properties and exhibits experimental neurotherapeutic benefits, but little is known about its in vivo effects on cerebral blood flow (CBF), functional evoked responses, and the associated changes in cerebral metabolic rate of oxygen (CMRO2). This study used magnetic resonance imaging (MRI) to evaluate the in vivo effects of a single intravenous MB therapeutic dose (0.5mg/kg) on basal CBF, blood oxygenation level-dependent (BOLD) and CBF responses to hypercapnic (5% CO2 in air) inhalation, as well as changes in BOLD, CBF, and CMRO2 during forepaw stimulation in the rat brain. MB did not have significant effects on arterial oxygen saturation, heart rate and fMRI responses to hypercapnia. However, MB significantly potentiated forepaw-evoked BOLD and CBF changes under normoxia. To further evaluate in vivo effects of MB under metabolic stress conditions, MRI measurements were also made under mild hypoxia (15% O2). Hypoxia per se increased evoked functional MRI responses. MB under hypoxia further potentiated forepaw-evoked BOLD, CBF and oxygen consumption responses relative to normoxia. These findings provide insights into MB's effects on cerebral hemodynamics in vivo and could help to optimize treatments in neurological diseases with mitochondrial dysfunction and oxidative stress.

Quantitative retinal and choroidal blood flow during light, dark adaptation and flicker light stimulation in rats using fluorescent microspheres.

PURPOSE: The present study aimed to quantify retinal and choroidal blood flow (BF) during light, dark adaptation and flicker light stimulation using the microsphere technique. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were anesthetized with isoflurane. Eyes were dark (Group I, n = 8), light (Group II, n = 8) adapted or stimulated with 10 Hz flicker light (Group III, n = 10). Retinal and choroidal BF were measured by a previously established method, using a mixture of 8 µm yellow-green and 10 µm red fluorescent microspheres. The microspheres were counted ex vivo in the dissected retina and choroid and in the reference arterial blood under a fluorescent microscope. RESULTS: The choroidal BF was 64.8 ± 29 µl/min (mean ± SD) during dark adaptation, not significantly different from that during light adaptation (66.0 ± 17.8 µl/min). The retinal BF was 13.5 ± 3.2 µl/min during 10 Hz flickering light stimulation, significantly higher than that during dark adaptation in the control fellow eyes (9.9 ± 2.9 µl/min). The choroidal BF values were not statistically different between flicker stimulation and dark adaptation. Retinal BF was 11.6 ± 2.9 µl/min during light adaptation. Dark adaptation did not increase retinal BF (Group I, 8.2 ± 2.4 µl/min; Group II, 9.9 ± 2.9 µl/min). CONCLUSIONS: These findings argue against a dark-induced or flicker-induced functional hyperemia in the choroid as a result of the demands of the outer retina. Retinal BF was not higher during dark adaptation. Our data support the conclusion that the inner retina has a higher energy demand in flicker conditions relative to dark.

Dopaminergic imaging of nonmotor manifestations in a rat model of Parkinson's disease by fMRI.

Nonmotor manifestations determine the life quality of patients with Parkinson's disease (PD). Identification of the nonmotor symptoms in PD as definite changes will represent a milestone in its diagnosis. Outcome measures that characterize nonmotor manifestations with specificity for dopaminergic deficiency are essential to that goal. Pain is a prevalent sensory disturbance in PD patients. The prevalence was reported to be up to 83%. Nociceptive stimuli under normal conditions elicit decreases in cerebral blood volume (CBV) in the striatum via dopaminergic neurotransmission. This nociception-induced CBV response is potentially to be defined as a characteristic of the pain symptom of PD. To validate this concept, steady-state CBV-weighted functional magnetic resonance imaging with iron oxide nanoparticles was employed to measure CBV changes in parkinsonian rats. Tyrosine hydroxylase immunohistology was used to identify the dopaminergic integrity to corroborate the imaging findings. Additional experiments that tested pain responses in parkinsonism were also carried out. The results revealed that the lesioned striatum exhibited a weakened CBV decrease in response to the nociceptive stimulus. This weakened CBV response occurred mainly in areas with dopaminergic denervation. A strong correspondence was observed between the distributions of the nociception-induced CBV responses and dopaminergic innervation. The persisting CBV signals in the striatum were abolished by the D2/D3 antagonist eticlopride. The findings of these behavioral, neuroimaging, immunohistological, and pharmacological experiments demonstrate that pain in a rat model of PD can be characterized by nociception induced striatal CBV signal changes with specificity for dopaminergic dysfunction.

Layer-specific manganese-enhanced MRI of the retina in light and dark adaptation.

PURPOSE: To employ functional manganese-enhanced MRI (MEMRI) to image layer-specific changes in calcium-dependent activities in the rat retina during light versus dark adaptation. METHODS: Functional MEMRI at 20 × 20 × 700 µm was used to study light and dark adaptation in the same animals (N = 10) in which one eye was covered and the fellow eye was not. The activity encoding of the light and dark adaptation was achieved in awake conditions and imaged under anesthesia. T(1)-weighted MRI at 11.7 tesla (T) was performed using two identical radiofrequency transceiver coils to allow interleaved MRI acquisitions of the two eyes. An intravascular contrast agent was also used to verify layer assignments. RESULTS: MEMRI detected contrasts among the inner retina, outer retina, and choroid. Independent confirmation of the vascular layers and boundaries between layers was documented with an intravascular contrast agent. The retinal layer thicknesses agreed with published data. The outer retina had lower MEMRI activity in light compared with dark adaption (P < 0.001), consistent with the increased metabolic demand associated with the "dark current." The inner retina had higher MEMRI activity in light compared with dark adaption (P < 0.05). The choroid MEMRI activity was not statistically different between light and dark adaptation (P > 0.05). CONCLUSIONS: This study demonstrated a high-resolution MEMRI protocol to image functional activities among different layers of the retinas in awake animals during light and dark adaptation. This approach could have potential applications in animal models of retinal dysfunction.

Layer-specific blood-flow MRI of retinitis pigmentosa in RCS rats.

The Royal College of Surgeons (RCS) rat is an established animal model of retinitis pigmentosa, a family of inherited retinal diseases which starts with loss of peripheral vision and progresses to eventual blindness. Blood flow (BF), an important physiological parameter, is intricately coupled to metabolic function under normal physiological conditions and is perturbed in many neurological and retinal diseases. This study reports non-invasive high-resolution MRI (44 × 44 × 600 µm) to image quantitative retinal and choroidal BF and layer-specific retinal thicknesses in RCS rat retinas at different stages of retinal degeneration compared with age-matched controls. The unique ability to separate retinal and choroidal BF was made possible by the depth-resolved MRI technique. RBF decreased with progressive retinal degeneration, but ChBF did not change in RCS rats up to post-natal day 90. We concluded that choroidal and retinal circulations have different susceptibility to progressive retinal degeneration in RCS rats. Layer-specific retinal thickness became progressively thinner and was corroborated by histological analysis in the same animals. MRI can detect progressive anatomical and BF changes during retinal degeneration with laminar resolution.

Investigation of the cerebral hemodynamic response function in single blood vessels by functional photoacoustic microscopy.

The specificity of the hemodynamic response function (HRF) is determined spatially by the vascular architecture and temporally by the evolution of hemodynamic changes. Here, we used functional photoacoustic microscopy (fPAM) to investigate single cerebral blood vessels of rats after left forepaw stimulation. In this system, we analyzed the spatiotemporal evolution of the HRFs of the total hemoglobin concentration (HbT), cerebral blood volume (CBV), and hemoglobin oxygen saturation (SO(2)). Changes in specific cerebral vessels corresponding to various electrical stimulation intensities and durations were bilaterally imaged with 36 × 65-µm(2) spatial resolution. Stimulation intensities of 1, 2, 6, and 10 mA were applied for periods of 5 or 15 s. Our results show that the relative functional changes in HbT, CBV, and SO(2) are highly dependent not only on the intensity of the stimulation, but also on its duration. Additionally, the duration of the stimulation has a strong influence on the spatiotemporal characteristics of the HRF as shorter stimuli elicit responses only in the local vasculature (smaller arterioles), whereas longer stimuli lead to greater vascular supply and drainage. This study suggests that the current fPAM system is reliable for studying relative cerebral hemodynamic changes, as well as for offering new insights into the dynamics of functional cerebral hemodynamic changes in small animals.

Transcranial imaging of functional cerebral hemodynamic changes in single blood vessels using in vivo photoacoustic microscopy.

Optical imaging of changes in total hemoglobin concentration (HbT), cerebral blood volume (CBV), and hemoglobin oxygen saturation (SO(2)) provides a means to investigate brain hemodynamic regulation. However, high-resolution transcranial imaging remains challenging. In this study, we applied a novel functional photoacoustic microscopy technique to probe the responses of single cortical vessels to left forepaw electrical stimulation in mice with intact skulls. Functional changes in HbT, CBV, and SO(2) in the superior sagittal sinus and different-sized arterioles from the anterior cerebral artery system were bilaterally imaged with unambiguous 36 × 65-µm(2) spatial resolution. In addition, an early decrease of SO(2) in single blood vessels during activation (i.e., 'the initial dip') was observed. Our results indicate that the initial dip occurred specifically in small arterioles of activated regions but not in large veins. This technique complements other existing imaging approaches for the investigation of the hemodynamic responses in single cerebral blood vessels.

Design, simulation and experimental validation of a novel flexible neural probe for deep brain stimulation and multichannel recording.

An implantable micromachined neural probe with multichannel electrode arrays for both neural signal recording and electrical stimulation was designed, simulated and experimentally validated for deep brain stimulation (DBS) applications. The developed probe has a rough three-dimensional microstructure on the electrode surface to maximize the electrode-tissue contact area. The flexible, polyimide-based microelectrode arrays were each composed of a long shaft (14.9 mm in length) and 16 electrodes (5 µm thick and with a diameter of 16 µm). The ability of these arrays to record and stimulate specific areas in a rat brain was evaluated. Moreover, we have developed a finite element model (FEM) applied to an electric field to evaluate the volume of tissue activated (VTA) by DBS as a function of the stimulation parameters. The signal-to-noise ratio ranged from 4.4 to 5 over a 50 day recording period, indicating that the laboratory-designed neural probe is reliable and may be used successfully for long-term recordings. The somatosensory evoked potential (SSEP) obtained by thalamic stimulations and in vivo electrode-electrolyte interface impedance measurements was stable for 50 days and demonstrated that the neural probe is feasible for long-term stimulation. A strongly linear (positive correlation) relationship was observed among the simulated VTA, the absolute value of the SSEP during the 200 ms post-stimulus period (ΣSSEP) and c-Fos expression, indicating that the simulated VTA has perfect sensitivity to predict the evoked responses (c-Fos expression). This laboratory-designed neural probe and its FEM simulation represent a simple, functionally effective technique for studying DBS and neural recordings in animal models.

High-resolution 3D MR microangiography of the rat ocular circulation.

PURPOSE: To develop high-spatial-resolution magnetic resonance (MR) microangiography techniques to image the rat ocular circulation. MATERIALS AND METHODS: Animal experiments were performed with institutional Animal Care Committee approval. MR microangiography (resolution, 84×84×84 µm or 42×42×84 µm) of the rat eye (eight rats) was performed by using a custom-made small circular surface coil with an 11.7-T MR unit before and after monocrystalline iron oxide nanoparticle (MION) injection. MR microangiography measurements were made during air, oxygen, and carbogen inhalation. From three-dimensional MR microangiography, the retina was virtually flattened to enable en face views of various retinal depths, including the retinal and choroidal vascular layers. Signal intensity changes within the retinal or choroidal arteries and veins associated with gas challenges were analyzed. Statistical analysis was performed by using paired t tests, with P<.05 considered to indicate a significant difference. Bonferroni correction was used to adjust for multiple comparisons. RESULTS: The central retinal artery, long posterior ciliary arteries, and choroidal vasculature could be distinguished on MR microangiograms of the eye. With MR microangiography, retinal arteries and veins could be distinguished on the basis of blood oxygen level-dependent contrast. Carbogen inhalation-enhanced MR microangiography signal intensity in both the retina (P=.001) and choroid (P=.027) compared with oxygen inhalation. Carbogen inhalation showed significantly higher signal intensity changes in the retinal arteries (P=.001, compared with oxygen inhalation), but not in the veins (P=.549). With MION administration, MR microangiography depicted retinal arterial vasoconstriction when the animals were breathing oxygen (P=.02, compared with animals breathing air). CONCLUSION: MR microangiography of the eye allows depth-resolved imaging of small angiographic details of the ocular circulation. This approach may prove useful in studying microvascular pathologic findings and neurovascular dysfunction in the eye and retina.

Endogenous opioid-dopamine neurotransmission underlie negative CBV fMRI signals.

Previous studies showed noxious unilateral forepaw electrical stimulation surprisingly evoked negative blood-oxygenation-level-dependent (BOLD), cerebral blood flow (CBF), and cerebral blood volume (CBV) fMRI responses in the bilateral striatum whereas the local neuronal spike and c-Fos activities increased. These negative responses are associated with vasoconstriction and appeared to override the increased hemodynamic responses that typically accompanied with increased neural activity. The current study aimed to investigate the role of µ-opioid system in modulating vasoconstriction in the striatum associated with noxious stimulation on a 4.7-Tesla MRI scanner. Specifically, we investigated: i) how morphine (a µ-opioid receptor agonist) affects the vasoconstriction in the bilateral striatum associated with noxious electrical forepaw stimulation in rats, and ii) how naloxone (an opioid receptor antagonist) and eticlopride (a dopamine D(2)/D(3) receptor antagonist) modulates the morphine effects onwards. Injection of morphine enhanced the negative striatal CBV responses to noxious stimulation. Sequential injection of naloxone in the same animals abolished the stimulus-evoked vasoconstriction. In a separate group of animals, injection of eticlopride following morphine also reduced the vasoconstriction. Our findings suggested that noxious stimulation endogenously activated opioid and dopamine receptors in the striatum and thus leading to vasoconstriction.

A rare case of coiling of the brachial artery: a description of the sonographic features.

We report the case of a patient with 360° coiling of the brachial artery that had been previously misdiagnosed as aneurysms of the right brachial artery on sonography performed at another institution. The previous misdiagnosis occurred due to sonographic pitfalls in gray-scale and color imaging, which led the operator to make a false interpretation. Knowledge of Doppler analysis and sonographic interpretation of arterial coiling may improve the diagnostic accuracy for this condition.

3D magnetic resonance microscopy of the ex vivo retina.

3D-MR microscopy at 11.7T and 20 × 20 × 57 µm resolution was performed on formalin-fixed rat eyes with: (I) no contrast agent and (II) Gadodiamide (Omniscan(®) ) added to the fixative. Group I data showed generally poor contrast among layers. Group II data showed markedly better lamina-specific contrast with the nerve fiber + ganglion cell layer and inner nuclear layer being hypointense, and the inner plexiform, outer plexiform, outer nuclear layer, and the segments being hyperintense. The signal-to-noise ratio in group II was higher than group I, consistent with Gadodiamide acting as a T(1) -contrast agent. All major retinal layers were assigned and their thicknesses quantified with corroboration by histology. MR microscopy allows nondestructive examination of valuable specimens and could have applications in disease and in vivo.

Pharmacological MRI of the choroid and retina: blood flow and BOLD responses during nitroprusside infusion.

Nitroprusside, a vasodilatory nitric oxide donor, is clinically used during vascular surgery and to lower blood pressure in acute hypertension. This article reports a novel application of blood flow (BF) and blood oxygenation level dependent (BOLD) MRI on an 11.7T scanner to image the rat chorioretinal BF and BOLD changes associated with graded nitroprusside infusion. At low doses (1 or 2 µg/kg/min), nitroprusside increased BF as expected but decreased BOLD signals, showing an intriguing BF-BOLD uncoupling. At high doses (3-5 µg/kg/min), nitroprusside decreased BF and markedly decreased BOLD signals. To our knowledge, this is the first pharmacological MRI application of the retina. This approach has potential to open up new avenues to study the drug-related hemodynamic functions and to evaluate the effects of novel therapeutic interventions on BOLD and BF in the normal and diseased retinas.

A low cost color visual stimulator for fMRI.

This low cost visual stimulator was developed for use in small animal imaging. The stimulator uses a single tri-color LED for each eye and can output red, green, or blue light or any combination of the three. When all three LED colors are illuminated at the same time achromatic light is the output. The stimulator is almost entirely implemented in software with only minimal electronics. The LEDs are controlled via the parallel port of a desktop computer. Flicker frequency, wavelength, intensity and waveform shape are under software control. The LEDs are coupled to fiber optic cables which run into the MRI scanner room leaving the LEDs and the power source in the control room. Calibration with a radiometer shows the light output to be very linear from zero to full intensity. The stimulator was used in fMRI visual stimulation studies performed on Sprague Dawley rats with an 11.7Tesla magnet. As the stimulator is software driven, modifications to accommodate other protocols and extensions for new functionality can be readily incorporated. With this in mind, the visual stimulator circuit diagram and software including source code are available upon request.