Minocycline attenuation of rat corpus callosum abnormality mediated by low-dose lipopolysaccharide-induced microglia activation.

BACKGROUND: Microglia are resident innate immune cells in the brain, and activation of these myeloid cells results in secretion of a variety of pro-inflammatory molecules, leading to the development of neurodegenerative disorders. Lipopolysaccharide (LPS) is a widely used experimental stimulant in microglia activation. We have previously shown that LPS produced microglia activation and evoked detectable functional abnormalities in rat corpus callosum (CC) in vitro. Here, we further validated the effects of low-dose LPS-induced microglia activation and resultant white matter abnormality in the CC in an animal model and examined its attenuation by an anti-inflammatory agent minocycline. METHODS: Twenty-four SD rats were divided randomly into three groups and intra-peritoneally injected daily with saline, LPS, and LPS + minocycline, respectively. All animals were subject to MRI tests 6 days post-injection. The animals were then sacrificed to harvest the CC tissues for electrophysiology, western blotting, and immunocytochemistry. One-way ANOVA with Tukey's post-test of all pair of columns was employed statistical analyses. RESULTS: Systemic administration of LPS produced microglial activation in the CC as illustrated by Iba-1 immunofluorescent staining. We observed that a large number of Iba-1-positive microglial cells were hyper-ramified with hypertrophic somata or even amoeba like in the LPS-treated animals, and such changes were significantly reduced by co-administration of minocycline. Electrophysiological recordings of axonal compound action potential (CAP) in the brain slices contained the CC revealed an impairment on the CC functionality as detected by a reduction in CAP magnitude. Such an impairment was supported by a reduction of fast axonal transportation evidenced by ß-amyloid precursor protein accumulation. These alterations were attenuated by minocycline, demonstrating minocycline reduction of microglia-mediated interruption of white matter integrity and function in the CC. CONCLUSIONS: Systemic administration of LPS produced microglia activation in the CC and resultant functional abnormalities that were attenuated by an anti-inflammatory agent minocycline.

Motor-Skill Learning Is Dependent on Astrocytic Activity.

Motor-skill learning induces changes in synaptic structure and function in the primary motor cortex through the involvement of a long-term potentiation- (LTP-) like mechanism. Although there is evidence that calcium-dependent release of gliotransmitters by astrocytes plays an important role in synaptic transmission and plasticity, the role of astrocytes in motor-skill learning is not known. To test the hypothesis that astrocytic activity is necessary for motor-skill learning, we perturbed astrocytic function using pharmacological and genetic approaches. We find that perturbation of astrocytes either by selectively attenuating IP3R2 mediated astrocyte Ca(2+) signaling or using an astrocyte specific metabolic inhibitor fluorocitrate (FC) results in impaired motor-skill learning of a forelimb reaching-task in mice. Moreover, the learning impairment caused by blocking astrocytic activity using FC was rescued by administration of the gliotransmitter D-serine. The learning impairments are likely caused by impaired LTP as FC blocked LTP in slices and prevented motor-skill training-induced increases in synaptic AMPA-type glutamate receptor in vivo. These results support the conclusion that normal astrocytic Ca(2+) signaling during a reaching task is necessary for motor-skill learning.

Compartmentalization of immune responses during Staphylococcus aureus cranial bone flap infection.

Decompressive craniectomy is often required after head trauma, stroke, or cranial bleeding to control subsequent brain swelling and prevent death. The infection rate after cranial bone flap replacement ranges from 0.8% to 15%, with an alarming frequency caused by methicillin-resistant Staphylococcus aureus, which is problematic because of recalcitrance to antibiotic therapy. Herein we report the establishment of a novel mouse model of S. aureus cranial bone flap infection that mimics several aspects of human disease. Bacteria colonized bone flaps for up to 4 months after infection, as revealed by scanning electron microscopy and quantitative culture, demonstrating the chronicity of the model. Analysis of a human cranial bone flap with confirmed S. aureus infection by scanning electron microscopy revealed similar structural attributes as the mouse model, demonstrating that it closely parallels structural facets of human disease. Inflammatory indices were most pronounced within the subcutaneous galeal compartment compared with the underlying brain parenchyma. Specifically, neutrophil influx and chemokine expression (CXCL2 and CCL5) were markedly elevated in the galea, which demonstrated substantial edema on magnetic resonance images, whereas the underlying brain parenchyma exhibited minimal involvement. Evaluation of immune mechanisms required for bacterial containment and inflammation revealed critical roles for MyD88-dependent signaling and neutrophils. This novel mouse model of cranial bone flap infection can be used to identify key immunologic and therapeutic mechanisms relevant to persistent bone flap infection in humans.

Mouse brain fixation to preserve In vivo manganese enhancement for ex vivo manganese-enhanced MRI.

PURPOSE: To develop a tissue fixation method that preserves in vivo manganese enhancement for ex vivo magnetic resonance imaging (MRI). The needs are clear, as conventional in vivo manganese-enhanced MRI (MEMRI) applied to live animals is time-limited, hence limited in spatial resolution and signal-to-noise ratio (SNR). Ex vivo applications can achieve superior spatial resolution and SNR through increased signal averaging and optimized radiofrequency coil designs. A tissue fixation method that preserves in vivo Mn(2+) enhancement postmortem is necessary for ex vivo MEMRI. MATERIALS AND METHODS: T1 measurements and T1 -weighted MRI were performed on MnCl2 -administered mice. The mice were then euthanized and the brains were fixed using one of two brain tissue fixation methods: aldehyde solution or focused beam microwave irradiation (FBMI). MRI was then performed on the fixed brains. RESULTS: T1 values and T1 -weighted signal contrasts were comparable between in vivo and ex vivo scans on aldehyde-fixed brains. FBMI resulted in the loss of Mn(2+) enhancement. CONCLUSION: Aldehyde fixation, not FBMI, maintained in vivo manganese enhancement for ex vivo MEMRI.

Loss of neuronal integrity during progressive HIV-1 infection of humanized mice.

Neuronal damage induced by ongoing human immunodeficiency virus type 1 (HIV-1) infection was investigated in humanized NOD/scid-IL-2Rγ(c)(null) mice transplanted at birth with human CD34-positive hematopoietic stem cells. Mice infected at 5 months of age and followed for up to 15 weeks maintained significant plasma viral loads and showed reduced numbers of CD4(+) T-cells. Prospective serial proton magnetic resonance spectroscopy tests showed selective reductions in cortical N-acetyl aspartate in infected animals. Diffusion tensor imaging revealed structural changes in cortical gray matter. Postmortem immunofluorescence brain tissue examinations for neuronal and glial markers, captured by multispectral imaging microscopy and quantified by morphometric and fluorescence emission, showed regional reduction of neuronal soma and synaptic architectures. This was evidenced by loss of microtubule-associated protein 2, synaptophysin, and neurofilament antigens. This study is the first, to our knowledge, demonstrating lost neuronal integrity after HIV-1 infection in humanized mice. As such, the model permits studies of the relationships between ongoing viral replication and virus-associated neurodegeneration.

Organic radical contrast agents for magnetic resonance imaging.

We report a molecular design that provides an intravenously injectable organic radical contrast agent (ORCA) for which the molecular (1)H water relaxivity (r(1)) is ca. 5 mM(-1) s(-1). The ORCA is based on spirocyclohexyl nitroxide radicals and poly(ethylene glycol) chains conjugated to a fourth-generation polypropylenimine dendrimer scaffold. The metal-free ORCA has a long shelf life and provides selectively enhanced magnetic resonance imaging in mice for over 1 h.

Correction to "Luteinizing Hormone Releasing Hormone-Targeted Cisplatin-Loaded Magnetite Nanoclusters for Simultaneous MR Imaging and Chemotherapy of Ovarian Cancer".

[This corrects the article PMC10317193.].

Nanoformulated Antiretroviral Therapy Attenuates Brain Metabolic Oxidative Stress.

Antiretroviral therapy (ART) restricts human immunodeficiency virus type one (HIV-1) replication and by so doing, improves the quality and longevity of life for infected people. Nonetheless, treatment can also lead to adverse clinical outcomes such as drug resistance and systemic adverse events. Both could be affected by long-acting slow effective release ART. Indeed, maintenance of sustained plasma drug levels, for weeks or months, after a single high-level dosing, could improve regimen adherence but, at the same time, affect systemic toxicities. Of these, the most troubling are those that affect the central nervous system (CNS). To address this, dolutegravir (Tivicay, DTG), a potent and durable HIV integrase inhibitor used effectively in combination ART was tested. Rodents were administered parenteral 45-mg/kg doses. DTG-associated changes in CNS homeostasis were assessed by measuring brain metabolic activities. After antiretroviral treatment, brain subregions were dissected and screened by mass spectrometry-based metabolomics. Metabolic drug-related dysregulation of energy and oxidative stress were readily observed within the cerebellum and frontal cortex following native drug administrations. Each was associated with alterations in neural homeostasis and depleted canonical oxidation protection pools that included glutathione and ascorbic acid. Surprisingly, the oxidative stress-related metabolites were completely attenuated when DTG was administered as nanoformulations. These data demonstrate the importance of formulation design in control of DTG or perhaps other antiretroviral drug-associated CNS events.

Ingress of blood-borne macrophages across the blood-brain barrier in murine HIV-1 encephalitis.

Blood-borne macrophage ingress into brain in HIV-1 associated neurocognitive disorders governs the tempo of disease. We used superparamagnetic iron-oxide particles loaded into murine bone marrow-derived macrophages (BMM) injected intravenously into HIV-1 encephalitis mice to quantitatively assess BMM entry into diseased brain regions. Magnetic resonance imaging tests were validated by histological coregistration and enhanced image processing. The demonstration of robust BMM migration into areas of focal encephalitis provide 'proof of concept' for the use of MRI to monitor macrophage ingress into brain.

MiR-133a Mimic Alleviates T1DM-Induced Systolic Dysfunction in Akita: An MRI-Based Study.

Diabetic cardiomyopathy is a leading cause of heart failure. Developing a novel therapeutic strategy for diabetic cardiomyopathy and characterizing animal models used for diabetes mellitus (DM) are important. Insulin 2 mutant (Ins2+/-) Akita is a spontaneous, genetic, mouse model for T1DM, which is relevant to humans. There are contrasting reports on systolic dysfunction and pathological remodeling (hypertrophy and fibrosis) in Akita heart. Here, we used magnetic resonance imaging (MRI) approach, a gold standard reference for evaluating cardiac function, to measure ejection fraction (indicator of systolic dysfunction) in Akita. Moreover, we performed Wheat Germ Agglutinin (WGA) and hematoxylin and Eosin stainings to determine cardiac hypertrophy, and Masson's Trichrome and picrosirius red stainings to determine cardiac fibrosis in Akita. MiR-133a, an anti-hypertrophy and anti-fibrosis miRNA, is downregulated in Akita heart. We determined if miR-133a mimic treatment could mitigate systolic dysfunction and remodeling in Akita heart. Our MRI results revealed decreased ejection fraction in Akita as compared to WT and increased ejection fraction in miR-133a mimic-treated Akita. We also found that miR-133a mimic treatment mitigates T1DM-induced cardiac hypertrophy and fibrosis in Akita. We conclude that Akita shows cardiac hypertrophy, fibrosis and systolic dysfunction and miR-133a mimic treatment to Akita could ameliorate them.

Multimodal Theranostic Nanoformulations Permit Magnetic Resonance Bioimaging of Antiretroviral Drug Particle Tissue-Cell Biodistribution.

RATIONALE: Long-acting slow effective release antiretroviral therapy (LASER ART) was developed to improve patient regimen adherence, prevent new infections, and facilitate drug delivery to human immunodeficiency virus cell and tissue reservoirs. In an effort to facilitate LASER ART development, "multimodal imaging theranostic nanoprobes" were created. These allow combined bioimaging, drug pharmacokinetics and tissue biodistribution tests in animal models. METHODS: Europium (Eu3+)- doped cobalt ferrite (CF) dolutegravir (DTG)- loaded (EuCF-DTG) nanoparticles were synthesized then fully characterized based on their size, shape and stability. These were then used as platforms for nanoformulated drug biodistribution. RESULTS: Folic acid (FA) decoration of EuCF-DTG (FA-EuCF-DTG) nanoparticles facilitated macrophage targeting and sped drug entry across cell barriers. Macrophage uptake was higher for FA-EuCF-DTG than EuCF-DTG nanoparticles with relaxivities of r2 = 546 mM-1s-1 and r2 = 564 mM-1s-1 in saline, and r2 = 850 mM-1s-1 and r2 = 876 mM-1s-1 in cells, respectively. The values were ten or more times higher than what was observed for ultrasmall superparamagnetic iron oxide particles (r2 = 31.15 mM-1s-1 in saline) using identical iron concentrations. Drug particles were detected in macrophage Rab compartments by dual fluorescence labeling. Replicate particles elicited sustained antiretroviral responses. After parenteral injection of FA-EuCF-DTG and EuCF-DTG into rats and rhesus macaques, drug, iron and cobalt levels, measured by LC-MS/MS, magnetic resonance imaging, and ICP-MS were coordinate. CONCLUSION: We posit that these theranostic nanoprobes can assess LASER ART drug delivery and be used as part of a precision nanomedicine therapeutic strategy.

Quantitative magnetic resonance and SPECT imaging for macrophage tissue migration and nanoformulated drug delivery.

We posit that the same mononuclear phagocytes (MP) [bone marrow (BM) and blood monocytes, tissue macrophages, microglia, and dendritic cells] which serve as targets, reservoirs, and vehicles for HIV dissemination, can be used as vehicles for antiretroviral therapy (ART). Toward this end, BM macrophages (BMM) were used as carriers for nanoparticle-formulated indinavir (NP-IDV), and the cell distribution was monitored by single photon emission computed tomography (SPECT), transverse relation time (T2)* weighted magnetic resonance imaging (MRI), histology, and gamma-scintillation spectrometry. BMM labeled with super paramagnetic iron oxide and/or 111indium oxine were infused i.v. into naïve mice. During the first 7 h, greater than 86% of cell label was recorded within the lungs. On Days 1, 3, 5, and 7, less than 10% of BMM were in lungs, and 74-81% and 13-18% were in liver and spleen, respectively. On a tissue volume basis, as determined by SPECT and MRI, BMM densities in spleen and liver were significantly greater than other tissues. Migration into the lymph nodes on Days 1 and 7 accounted for 1.5-2% of the total BMM. Adoptive transfer of BMM loaded with NP-IDV produced drug levels in lymphoid and nonlymphoid tissues that exceeded reported therapeutic concentrations by 200- to 350-fold on Day 1 and remained in excess of 100- to 300-fold on Day 14. These data show real-time kinetics and destinations of macrophage trafficking and demonstrate the feasibility of monitoring macrophage-based, nanoformulated ART.

MEMRI is a biomarker defining nicotine-specific neuronal responses in subregions of the rodent brain.

Nicotine dependence is defined by dopaminergic neuronal activation within the nucleus accumbens (ACB) and by affected neural projections from nicotine-stimulated neurons. Control of any subsequent neural activities would underpin any smoking cessation strategy. While extensive efforts have been made to study the pathophysiology of nicotine addiction, more limited works were developed to find imaging biomarkers. If such biomarkers are made available, addictive behaviors could be monitored noninvasively. To such ends, we employed manganese (Mn2+)-enhanced magnetic resonance imaging (MEMRI) to determine whether it could be used to monitor neuronal activities after acute and chronic nicotine exposure in rats. The following were observed. Mn2+ infusion identified ACB and hippocampal (HIP) neuronal activities following acute nicotine administration. Chronic exposure was achieved by week long subcutaneously implanted nicotine mini-pump. Here nicotine was shown to activate neurons in the ACB, HIP, and the prefrontal and insular cortex. These are all central nervous system reward regions linked to drug addiction. In conclusion, MEMRI is demonstrated to be a powerful imaging tool to study brain subregion specific neuronal activities affected by nicotine. Thus, we posit that MEMRI could be used to assess smoking-associated tolerance, withdrawal and as such serve as a pre-clinical screening tool for addiction cessation strategies in humans.

Development of europium doped core-shell silica cobalt ferrite functionalized nanoparticles for magnetic resonance imaging.

The size, shape and chemical composition of europium (Eu3+) cobalt ferrite (CFEu) nanoparticles were optimized for use as a "multimodal imaging nanoprobe" for combined fluorescence and magnetic resonance bioimaging. Doping Eu3+ ions into a CF structure imparts unique bioimaging and magnetic properties to the nanostructure that can be used for real-time screening of targeted nanoformulations for tissue biodistribution assessment. The CFEu nanoparticles (size ∼7.2nm) were prepared by solvothermal techniques and encapsulated into poloxamer 407-coated mesoporous silica (Si-P407) to form superparamagnetic monodisperse Si-CFEu nanoparticles with a size of ∼140nm. Folic acid (FA) nanoparticle decoration (FA-Si-CFEu, size ∼140nm) facilitated monocyte-derived macrophage (MDM) targeting. FA-Si-CFEu MDM uptake and retention was higher than seen with Si-CFEu nanoparticles. The transverse relaxivity of both Si-CFEu and FA-Si-CFEu particles were r2=433.42mM-1s-1 and r2=419.52mM-1s-1 (in saline) and r2=736.57mM-1s-1 and r2=814.41mM-1s-1 (in MDM), respectively. The results were greater than a log order-of-magnitude than what was observed at replicate iron concentrations for ultrasmall superparamagnetic iron oxide (USPIO) particles (r2=31.15mM-1s-1 in saline) and paralleled data sets obtained for T2 magnetic resonance imaging. We now provide a developmental opportunity to employ these novel particles for theranostic drug distribution and efficacy evaluations. STATEMENT OF SIGNIFICANCE: A novel europium (Eu3+) doped cobalt ferrite (Si-CFEu) nanoparticle was produced for use as a bioimaging probe. Its notable multifunctional, fluorescence and imaging properties, allows rapid screening of future drug biodistribution. Decoration of the Si-CFEu particles with folic acid increased its sensitivity and specificity for magnetic resonance imaging over a more conventional ultrasmall superparamagnetic iron oxide particles. The future use of these particles in theranostic tests will serve as a platform for designing improved drug delivery strategies to combat inflammatory and infectious diseases.

Nitroxide-Based Macromolecular Contrast Agents with Unprecedented Transverse Relaxivity and Stability for Magnetic Resonance Imaging of Tumors.

Metal-free magnetic resonance imaging (MRI) agents could overcome the established toxicity associated with metal-based agents in some patient populations and enable new modes of functional MRI in vivo. Herein, we report nitroxide-functionalized brush-arm star polymer organic radical contrast agents (BASP-ORCAs) that overcome the low contrast and poor in vivo stability associated with nitroxide-based MRI contrast agents. As a consequence of their unique nanoarchitectures, BASP-ORCAs possess per-nitroxide transverse relaxivities up to ∼44-fold greater than common nitroxides, exceptional stability in highly reducing environments, and low toxicity. These features combine to provide for accumulation of a sufficient concentration of BASP-ORCA in murine subcutaneous tumors up to 20 h following systemic administration such that MRI contrast on par with metal-based agents is observed. BASP-ORCAs are, to our knowledge, the first nitroxide MRI contrast agents capable of tumor imaging over long time periods using clinical high-field 1H MRI techniques.

Quantitative 1H magnetic resonance spectroscopic imaging determines therapeutic immunization efficacy in an animal model of Parkinson's disease.

Nigrostriatal degeneration, the pathological hallmark of Parkinson's disease (PD), is mirrored by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication. MPTP-treated animals show the common behavioral, motor, and pathological features of human disease. We demonstrated previously that adoptive transfer of Copaxone (Cop-1) immune cells protected the nigrostriatal dopaminergic pathway in MPTP-intoxicated mice. Herein, we evaluated this protection by quantitative proton magnetic resonance spectroscopic imaging (1H MRSI). 1H MRSI performed in MPTP-treated mice demonstrated that N-acetyl aspartate (NAA) was significantly diminished in the substantia nigra pars compacta (SNpc) and striatum, regions most affected in human disease. When the same regions were coregistered with immunohistochemical stains for tyrosine hydroxylase, numbers of neuronal bodies and termini were similarly diminished. MPTP-intoxicated animals that received Cop-1 immune cells showed NAA levels, in the SNpc and striatum, nearly equivalent to PBS-treated animals. Moreover, adoptive transfer of immune cells from ovalbumin-immunized to MPTP-treated mice failed to alter NAA levels or protect dopaminergic neurons and their projections. These results demonstrate that 1H MRSI can evaluate dopaminergic degeneration and its protection by Cop-1 immunization strategies. Most importantly, the results provide a monitoring system to assess therapeutic outcomes for PD.

Neuroinflammation, Oxidative Stress and the Pathogenesis of Parkinson's Disease.

Neuroinflammatory processes play a significant role in the pathogenesis of Parkinson's disease (PD). Epidemiologic, animal, human, and therapeutic studies all support the presence of an neuroinflammatory cascade in disease. This is highlighted by the neurotoxic potential of microglia . In steady state, microglia serve to protect the nervous system by acting as debris scavengers, killers of microbial pathogens, and regulators of innate and adaptive immune responses. In neurodegenerative diseases, activated microglia affect neuronal injury and death through production of glutamate, pro-inflammatory factors, reactive oxygen species, quinolinic acid amongst others and by mobilization of adaptive immune responses and cell chemotaxis leading to transendothelial migration of immunocytes across the blood-brain barrier and perpetuation of neural damage. As disease progresses, inflammatory secretions engage neighboring glial cells, including astrocytes and endothelial cells, resulting in a vicious cycle of autocrine and paracrine amplification of inflammation perpetuating tissue injury. Such pathogenic processes contribute to neurodegeneration in PD. Research from others and our own laboratories seek to harness such inflammatory processes with the singular goal of developing therapeutic interventions that positively affect the tempo and progression of human disease.

Early Expression of Parkinson's Disease-Related Mitochondrial Abnormalities in PINK1 Knockout Rats.

PTEN-induced kinase 1 (PINK1) mutations are responsible for an autosomal recessive, familial form of Parkinson's disease. PINK1 protein is a Ser/Thr kinase localized to the mitochondrial membrane and is involved in many processes including mitochondrial trafficking, mitophagy, and proteasomal function. Using a new PINK1 knockout (PINK1 KO) rat model, we found altered brain metabolomic markers using magnetic resonance spectroscopy, identified changes in mitochondrial pathways with quantitative proteomics using sequential window acquisition of all theoretical spectra (SWATH) mass spectrometry, and demonstrated mitochondrial functional alterations through measurement of oxygen consumption and acidification rates. The observed alterations included reduced creatine, decreased levels of complex I of the electron transport chain, and increased proton leak in the electron transport chain in PINK1 KO rat brains. In conjunction, these results demonstrate metabolomic and mitochondrial alterations occur during the asymptomatic phase of Parkinson's disease in this model. These results indicate both potential early diagnostic markers and therapeutic pathways that can be used in PD.

Manganese-Enhanced Magnetic Resonance Imaging Reflects Brain Pathology During Progressive HIV-1 Infection of Humanized Mice.

Progressive human immunodeficiency viral (HIV) infection commonly leads to a constellation of cognitive, motor, and behavioral impairments. These are collectively termed HIV-associated neurocognitive disorders (HAND). While antiretroviral therapy (ART) reduces HAND severity, it does not affect disease prevalence. Despite decades of research, there remain no biomarkers for HAND and all potential comorbid conditions must first be excluded for a diagnosis to be made. To this end, we now report that manganese (Mn(2+))-enhanced magnetic resonance imaging (MEMRI) can reflect brain region-specific HIV-1-induced neuropathology in chronically virus-infected NOD/scid-IL-2Rγc(null) humanized mice. MEMRI diagnostics mirrors the abilities of Mn(2+) to enter and accumulate in affected neurons during disease. T1 relaxivity and its weighted signal intensity are proportional to Mn(2+) activities in neurons. In 16-week virus-infected humanized mice, altered MEMRI signal enhancement was easily observed in affected brain regions. These included, but were not limited to, the hippocampus, amygdala, thalamus, globus pallidus, caudoputamen, substantia nigra, and cerebellum. MEMRI signal was coordinated with levels of HIV-1 infection, neuroinflammation (astro- and micro-gliosis), and neuronal injury. MEMRI accurately demonstrates the complexities of HIV-1-associated neuropathology in rodents that reflects, in measure, the clinical manifestations of neuroAIDS as it is seen in a human host.