RESUMO
The precise mechanisms underlying the beneficial effects of regulatory T (Treg) cells on long-term tissue repair remain elusive. Here, using single-cell RNA sequencing and flow cytometry, we found that Treg cells infiltrated the brain 1 to 5 weeks after experimental stroke in mice. Selective depletion of Treg cells diminished oligodendrogenesis, white matter repair, and functional recovery after stroke. Transcriptomic analyses revealed potent immunomodulatory effects of brain-infiltrating Treg cells on other immune cells, including monocyte-lineage cells. Microglia depletion, but not T cell lymphopenia, mitigated the beneficial effects of transferred Treg cells on white matter regeneration. Mechanistically, Treg cell-derived osteopontin acted through integrin receptors on microglia to enhance microglial reparative activity, consequently promoting oligodendrogenesis and white matter repair. Increasing Treg cell numbers by delivering IL-2:IL-2 antibody complexes after stroke improved white matter integrity and rescued neurological functions over the long term. These findings reveal Treg cells as a neurorestorative target for stroke recovery.
Assuntos
Isquemia Encefálica/imunologia , AVC Isquêmico/imunologia , Microglia/imunologia , Osteopontina/imunologia , Recuperação de Função Fisiológica/imunologia , Linfócitos T Reguladores/imunologia , Substância Branca/imunologia , Animais , Modelos Animais de Doenças , Interleucina-2/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Bilateral common carotid artery (CCA) stenosis (BCAS) is a useful model to mimic vascular cognitive impairment and dementia (VCID). However, current BCAS models have the disadvantages of high cost and incompatibility with magnetic resonance imaging (MRI) scanning because of metal implantation. We have established a new low-cost VCID model that better mimics human VCID and is compatible with live-animal MRI. The right and the left CCAs were temporarily ligated to 32- and 34-gauge needles with three ligations, respectively. After needle removal, CCA blood flow, cerebral blood flow, white matter injury (WMI) and cognitive function were measured. In male mice, needle removal led to â¼49.8% and â¼28.2% blood flow recovery in the right and left CCA, respectively. This model caused persistent and long-term cerebral hypoperfusion in both hemispheres (more severe in the left hemisphere), and WMI and cognitive dysfunction in â¼90% of mice, which is more reliable compared with other models. Importantly, these pathologic changes and cognitive impairments lasted for up to 24 weeks after surgery. The survival rate over 24 weeks was 81.6%. Female mice showed similar cognitive dysfunction, but a higher survival rate (91.6%) and relatively milder white matter injury. A novel, low-cost VCID model compatible with live-animal MRI with long-term outcomes was established.SIGNIFICANCE STATEMENT Bilateral common carotid artery (CCA) stenosis (BCAS) is an animal model mimicking carotid artery stenosis to study vascular cognitive impairment and dementia (VCID). However, current BCAS models have the disadvantages of high cost and incompatibility with magnetic resonance imaging (MRI) scanning due to metal implantation. We established a new asymmetric BCAS model by ligating the CCA to various needle gauges followed by an immediate needle removal. Needle removal led to moderate stenosis in the right CCA and severe stenosis in the left CCA. This needle model replicates the hallmarks of VCID well in â¼90% of mice, which is more reliable compared with other models, has ultra-low cost, and is compatible with MRI scanning in live animals. It will provide a new valuable tool and offer new insights for VCID research.
Assuntos
Disfunção Cognitiva , Demência Vascular , Masculino , Camundongos , Feminino , Humanos , Animais , Constrição Patológica/complicações , Disfunção Cognitiva/etiologia , Modelos Animais de Doenças , Demência Vascular/diagnóstico por imagem , Demência Vascular/etiologia , Demência Vascular/patologia , Cognição , Camundongos Endogâmicos C57BLRESUMO
The connectivity of the hippocampus is essential to its functions. To gain a whole system view of intrahippocampal connectivity, ex vivo mesoscale (100 µm isotropic resolution) multi-shell diffusion MRI (11.7T) and tractography were performed on entire post-mortem human right hippocampi. Volumetric measurements indicated that the head region was largest followed by the body and tail regions. A unique anatomical organization in the head region reflected a complex organization of the granule cell layer (GCL) of the dentate gyrus. Tractography revealed the volumetric distribution of the perforant path, including both the tri-synaptic and temporoammonic pathways, as well as other well-established canonical connections, such as Schaffer collaterals. Visualization of the perforant path provided a means to verify the borders between the pro-subiculum and CA1, as well as between CA1/CA2. A specific angularity of different layers of fibers in the alveus was evident across the whole sample and allowed a separation of afferent and efferent connections based on their origin (i.e. entorhinal cortex) or destination (i.e. fimbria) using a cluster analysis of streamlines. Non-canonical translamellar connections running along the anterior-posterior axis were also discerned in the hilus. In line with "dentations" of the GCL, mossy fibers were bunching together in the sagittal plane revealing a unique lamellar organization and connections between these. In the head region, mossy fibers projected to the origin of the fimbria, which was distinct from the body and tail region. Mesoscale tractography provides an unprecedented systems view of intrahippocampal connections that underpin cognitive and emotional processing.
Assuntos
Hipocampo , Via Perfurante , Humanos , Hipocampo/diagnóstico por imagem , Córtex Entorrinal , Encéfalo , Imagem de Difusão por Ressonância MagnéticaRESUMO
Recent research highlights the function of regulatory T cells (Tregs) in white matter integrity in CNS diseases. Approaches that expand the number of Tregs have been utilized to improve stroke recovery. However, it remains unclear if Treg augmentation preserves white matter integrity early after stroke or promotes white matter repair. This study evaluates the effect of Treg augmentation on white matter injury and repair after stroke. Adult male C57/BL6 mice randomly received Treg or splenocyte (2 million, iv) transfer 2 h after transient (60 min) middle cerebral artery occlusion (tMCAO). Immunostaining showed improved white matter recovery after tMCAO in Treg-treated mice compared to mice received splenocytes. In another group of mice, IL-2/IL-2 antibody complexes (IL-2/IL-2Ab) or isotype IgG were administered (i.p) for 3 consecutive days starting 6 h after tMCAO, and repeated on day 10, 20 and 30. The IL-2/IL-2Ab treatment boosted the number of Tregs in blood and spleen and increased Treg infiltration into the ischemic brain. Longitudinal in vivo and ex vivo diffusion tensor imaging analysis revealed an increase in fractional anisotropy 28d and 35d, but not 14d, after stroke in IL-2/IL-2Ab-treated mice compared to isotype-treated mice, suggesting a delayed improvement in white matter integrity. IL-2/IL-2Ab also improved sensorimotor functions (rotarod test and adhesive removal test) 35d after stroke. There were correlations between white matter integrity and behavior performance. Immunostaining confirmed the beneficial effects of IL-2/IL-2Ab on white matter structures 35d after tMCAO. IL-2/IL-2Ab treatment starting as late as 5d after stroke still improved white matter integrity 21d after tMCAO, suggesting long-term salutary effects of Tregs on the late-stage tissue repair. We also found that IL-2/IL-2Ab treatment reduced the number of dead/dying OPCs and oligodendrocytes in the brain 3d after tMCAO. To confirm the direct effect of Tregs on remyelination, Tregs were cocultured with lysophosphatidyl choline (LPC)-treated organotypic cerebella. LPC exposure for 17 h induced demyelination in organotypic cultures, followed by gradual spontaneous remyelination upon removal of LPC. Co-culture with Tregs accelerated remyelination in organotypic cultures 7d after LPC. In conclusion, Boosting the number of Tregs protects oligodendrocyte lineage cells early after stroke and promotes long-term white matter repair and functional recovery. IL-2/IL-2Ab represents a feasible approach of Treg expansion for stroke treatment.
Assuntos
Acidente Vascular Cerebral , Substância Branca , Camundongos , Masculino , Animais , Linfócitos T Reguladores , Imagem de Tensor de Difusão , Interleucina-2/farmacologia , Camundongos Endogâmicos C57BLRESUMO
Differential microglial inflammatory responses play a role in regulation of differentiation and maturation of oligodendrocytes (OLs) in brain white matter. How microglia-OL crosstalk is altered by traumatic brain injury (TBI) and its impact on axonal myelination and neurological function impairment remain poorly understood. In this study, we investigated roles of a Na+/H+ exchanger (NHE1), an essential microglial pH regulatory protein, in microglial proinflammatory activation and OL survival and differentiation in a murine TBI model induced by controlled cortical impact. Similar TBI-induced contusion volumes were detected in the Cx3cr1-CreERT2 control (Ctrl) mice and selective microglial Nhe1 knockout (Cx3cr1-CreERT2;Nhe1flox/flox, Nhe1 cKO) mice. Compared to the Ctrl mice, the Nhe1 cKO mice displayed increased resistance to initial TBI-induced white matter damage and accelerated chronic phase of OL regeneration at 30 days post-TBI. The cKO brains presented increased anti-inflammatory phenotypes of microglia and infiltrated myeloid cells, with reduced proinflammatory transcriptome profiles. Moreover, the cKO mice exhibited accelerated post-TBI sensorimotor and cognitive functional recovery than the Ctrl mice. These phenotypic outcomes in cKO mice were recapitulated in C57BL6J wild-type TBI mice receiving treatment of a potent NHE1 inhibitor HOE642 for 1-7 days post-TBI. Taken together, these findings collectively demonstrated that blocking NHE1 protein stimulates restorative microglial activation in oligodendrogenesis and neuroprotection, which contributes to accelerated brain repair and neurological function recovery after TBI.
Assuntos
Lesões Encefálicas Traumáticas , Substância Branca , Animais , Lesões Encefálicas Traumáticas/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Oligodendroglia , Recuperação de Função FisiológicaRESUMO
The repair of white matter damage is of paramount importance for functional recovery after brain injuries. Here, we report that interleukin-4 (IL-4) promotes oligodendrocyte regeneration and remyelination. IL-4 receptor expression was detected in a variety of glial cells after ischemic brain injury, including oligodendrocyte lineage cells. IL-4 deficiency in knockout mice resulted in greater deterioration of white matter over 14 d after stroke. Consistent with these findings, intranasal delivery of IL-4 nanoparticles after stroke improved white matter integrity and attenuated long-term sensorimotor and cognitive deficits in wild-type mice, as revealed by histological immunostaining, electron microscopy, diffusion tensor imaging, and electrophysiology. The selective effect of IL-4 on remyelination was verified in an ex vivo organotypic model of demyelination. By leveraging primary oligodendrocyte progenitor cells (OPCs), microglia-depleted mice, and conditional OPC-specific peroxisome proliferator-activated receptor gamma (PPARγ) knockout mice, we discovered a direct salutary effect of IL-4 on oligodendrocyte differentiation that was mediated by the PPARγ axis. Our findings reveal a new regenerative role of IL-4 in the central nervous system (CNS), which lies beyond its known immunoregulatory functions on microglia/macrophages or peripheral lymphocytes. Therefore, intranasal IL-4 delivery may represent a novel therapeutic strategy to improve white matter integrity in stroke and other brain injuries.
Assuntos
Interleucina-4/metabolismo , Oligodendroglia/metabolismo , PPAR gama/metabolismo , Animais , Lesões Encefálicas , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatologia , Diferenciação Celular/fisiologia , Doenças Desmielinizantes/metabolismo , Interleucina-4/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/metabolismo , Bainha de Mielina/metabolismo , Regeneração Nervosa , Neurogênese , Oligodendroglia/fisiologia , PPAR gama/fisiologia , Recuperação de Função Fisiológica , Remielinização/fisiologia , Transdução de Sinais , Acidente Vascular Cerebral , Substância BrancaRESUMO
Persistent endoplasmic reticulum (ER) stress in neurons is associated with activation of inflammatory cells and subsequent neuroinflammation following traumatic brain injury (TBI); however, the underlying mechanism remains elusive. We found that induction of neuronal-ER stress, which was mostly characterized by an increase in phosphorylation of a protein kinase R-like ER kinase (PERK) leads to release of excess interferon (IFN)ß due to atypical activation of the neuronal-STING signaling pathway. IFNß enforced activation and polarization of the primary microglial cells to inflammatory M1 phenotype with the secretion of a proinflammatory chemokine CXCL10 due to activation of STAT1 signaling. The secreted CXCL10, in turn, stimulated the T-cell infiltration by serving as the ligand and chemoattractant for CXCR3+ T-helper 1 (Th1) cells. The activation of microglial cells and infiltration of Th1 cells resulted in white matter injury, characterized by impaired myelin basic protein and neurofilament NF200, the reduced thickness of corpus callosum and external capsule, and decline of mature oligodendrocytes and oligodendrocyte precursor cells. Intranasal delivery of CXCL10 siRNA blocked Th1 infiltration but did not fully rescue microglial activation and white matter injury after TBI. However, impeding PERK-phosphorylation through the administration of GSK2656157 abrogated neuronal induction of IFNß, switched microglial polarization to M2 phenotype, prevented Th1 infiltration, and increased Th2 and Treg levels. These events ultimately attenuated the white matter injury and improved anxiety and depressive-like behavior following TBI.SIGNIFICANCE STATEMENT A recent clinical study showed that human brain trauma patients had enhanced expression of type-1 IFN; suggests that type-1 IFN signaling may potentially influence clinical outcome in TBI patients. However, it was not understood how TBI leads to an increase in IFNß and whether induction of IFNß has any influence on neuroinflammation, which is the primary reason for morbidity and mortality in TBI. Our study suggests that induction of PERK phosphorylation, a characteristic feature of ER stress is responsible for an increase in neuronal IFNß, which, in turn, activates microglial cells and subsequently manifests the infiltration of T cells to induce neuroinflammation and subsequently white matter injury. Blocking PERK phosphorylation using GSK2656157 (or PERK knockdown) the whole cascade of neuroinflammation was attenuated and improved cognitive function after TBI.
Assuntos
Lesões Encefálicas Traumáticas/fisiopatologia , Estresse do Retículo Endoplasmático/fisiologia , Microglia/metabolismo , Linfócitos T , Substância Branca/fisiopatologia , eIF-2 Quinase/metabolismo , Animais , Feminino , Interferon beta/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Substância Branca/lesõesRESUMO
BACKGROUND: Chronic cerebral hypoperfusion (CCH) causes white matter damage and cognitive impairment, in which astrogliosis is the major pathology. However, underlying cellular mechanisms are not well defined. Activation of Na+/H+ exchanger-1 (NHE1) in reactive astrocytes causes astrocytic hypertrophy and swelling. In this study, we examined the role of NHE1 protein in astrogliosis, white matter demyelination, and cognitive function in a murine CCH model with bilateral carotid artery stenosis (BCAS). METHODS: Sham, BCAS, or BCAS mice receiving vehicle or a selective NHE1 inhibitor HOE642 were monitored for changes of the regional cerebral blood flow and behavioral performance for 28 days. Ex vivo MRI-DTI was subsequently conducted to detect brain injury and demyelination. Astrogliosis and demyelination were further examined by immunofluorescence staining. Astrocytic transcriptional profiles were analyzed with bulk RNA-sequencing and RT-qPCR. RESULTS: Chronic cerebral blood flow reduction and spatial working memory deficits were detected in the BCAS mice, along with significantly reduced mean fractional anisotropy (FA) values in the corpus callosum, external capsule, and hippocampus in MRI DTI analysis. Compared with the sham control mice, the BCAS mice displayed demyelination and axonal damage and increased GFAP+ astrocytes and Iba1+ microglia. Pharmacological inhibition of NHE1 protein with its inhibitor HOE642 prevented the BCAS-induced gliosis, damage of white matter tracts and hippocampus, and significantly improved cognitive performance. Transcriptome and immunostaining analysis further revealed that NHE1 inhibition specifically attenuated pro-inflammatory pathways and NADPH oxidase activation. CONCLUSION: Our study demonstrates that NHE1 protein is involved in astrogliosis with pro-inflammatory transformation induced by CCH, and its blockade has potentials for reducing astrogliosis, demyelination, and cognitive impairment.
Assuntos
Astrócitos/efeitos dos fármacos , Estenose das Carótidas/tratamento farmacológico , Cognição/efeitos dos fármacos , Gliose/tratamento farmacológico , Guanidinas/uso terapêutico , Sulfonas/uso terapêutico , Substância Branca/efeitos dos fármacos , Animais , Astrócitos/patologia , Estenose das Carótidas/patologia , Circulação Cerebrovascular/efeitos dos fármacos , Disfunção Cognitiva/patologia , Gliose/patologia , Guanidinas/farmacologia , Inflamação/patologia , Camundongos , Microglia/efeitos dos fármacos , Microglia/patologia , Trocador 1 de Sódio-Hidrogênio/antagonistas & inibidores , Sulfonas/farmacologia , Substância Branca/patologiaRESUMO
Recombinant tissue plasminogen activator (tPA) is a Food and Drug Administration-approved thrombolytic treatment for ischemic stroke. tPA is also naturally expressed in glial and neuronal cells of the brain, where it promotes axon outgrowth and synaptic plasticity. However, there are conflicting reports of harmful versus neuroprotective effects of tPA in acute brain injury models. Furthermore, its impact on white matter integrity in preclinical traumatic brain injury (TBI) has not been thoroughly explored, although white matter disruption is a better predictor of long-term clinical outcomes than focal lesion volumes. Here we show that the absence of endogenous tPA in knockout mice impedes long-term recovery of white matter and neurological function after TBI. tPA-knockout mice exhibited greater asymmetries in forepaw use, poorer sensorimotor balance and coordination, and inferior spatial learning and memory up to 35 d after TBI. White matter damage was also more prominent in tPA knockouts, as shown by diffusion tensor imaging, histological criteria, and electrophysiological assessments of axon conduction properties. Replenishment of tPA through intranasal application of the recombinant protein in tPA-knockout mice enhanced neurological function, the structural and functional integrity of white matter, and postinjury compensatory sprouting in corticofugal projections. tPA also promoted neurite outgrowth in vitro, partly through the epidermal growth factor receptor. Both endogenous and exogenous tPA protected against white matter injury after TBI without increasing intracerebral hemorrhage volumes. These results unveil a previously unappreciated role for tPA in the protection and/or repair of white matter and long-term functional recovery after TBI.
Assuntos
Lesões Encefálicas Traumáticas/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Ativador de Plasminogênio Tecidual/uso terapêutico , Substância Branca/efeitos dos fármacos , Animais , Lesões Encefálicas Traumáticas/patologia , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Nervosas/efeitos dos fármacos , Rede Nervosa/efeitos dos fármacos , Proteínas Recombinantes , Substância Branca/patologiaRESUMO
To assess whether quantitative T1 relaxometry can measure permeability, chronic inflammation and mural thickening of mouse bladder wall. Adult female C57BL6 mice unexposed to radiation (controls) or 40 wk postirradiation of 10 Gy were scanned at 9.4 T before and after instillation (0.1 mL) of aqueous, novel contrast mixture (NCM) containing 4 mM gadobutrol and 5 mM ferumoxytol. Rapid acquisition with refocused echo (RARE) sequence was used with variable repetition times (TR). Pixel-wise maps of T1 relaxation times for the segmented bladder wall layers were generated from voxel-wise, nonlinear least square data fitting of TR-dependent signal intensity acquired with TR array of 0.4-10 s followed by the histology of harvested bladder. Significant differences between precontrast and postcontrast T1 (ΔT1) were noted in urothelium and lamina propria of both groups but only in detrusor of irradiated group (P < 0.001; 2-way ANOVA). Nearly twofold higher gadobutrol permeability (550 ± 73 vs. 294 ± 160 µM; P < 0.01) derived as per 1/ΔT1 = r1. [C] in urothelium of irradiated group. Inflammation and bladder wall thickening (0.75 ± 0. vs. 0.44 ± 0.08 mm; P < 0.001) predicted by MRI was subsequently confirmed by histology and altered expression of CD45 and zonula occludens-1 (ZO-1) relative to controls. NCM enhanced MRI relies on the retention of large molecular weight ferumoxytol in lumen for negative contrast, while permeation of the non-ionic, small molecular weight gadobutrol through ZO-1 generates positive contrast in bladder wall for virtual measurement of paracellular permeability and assessment of chronic inflammation in thin and distensible bladder wall, which is also defined by its variable shape and location within pelvis.
Assuntos
Inflamação/diagnóstico por imagem , Doenças da Bexiga Urinária/diagnóstico por imagem , Animais , Meios de Contraste/farmacocinética , Meios de Contraste/farmacologia , Feminino , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Compostos Organometálicos/farmacocinética , Compostos Organometálicos/farmacologia , Permeabilidade , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/fisiologia , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
The role of hippocampal connectivity in mesial temporal lobe epilepsy (mTLE) remains poorly understood. The use of ex vivo hippocampal samples excised from patients with mTLE affords mesoscale diffusion magnetic resonance imaging (MRI) to identify individual cell layers, such as the pyramidal (PCL) and granule cell layers (GCL), which are thought to be impacted by seizure activity. Diffusion tensor imaging (DTI) of control (n = 3) and mTLE (n = 7) hippocampi on an 11.7 T MRI scanner allowed us to reveal intra-hippocampal connectivity and evaluate how epilepsy affected mean (MD), axial (AD), and radial diffusivity (RD), as well as fractional anisotropy (FA). Regional measurements indicated a volume loss in the PCL of the cornu ammonis (CA) 1 subfield in mTLE patients compared to controls, which provided anatomical context. Diffusion measurements, as well as streamline density, were generally higher in mTLE patients compared to controls, potentially reflecting differences due to tissue fixation. mTLE measurements were more variable than controls. This variability was associated with disease severity, as indicated by a strong correlation (r = 0.87) between FA in the stratum radiatum and the frequency of seizures in patients. MD and RD of the PCL in subfields CA3 and CA4 also correlated strongly with disease severity. No correlation of MR measures with disease duration was evident. These results reveal the potential of mesoscale diffusion MRI to examine layer-specific diffusion changes and connectivity to determine how these relate to clinical measures. Improving the visualization of intra-hippocampal connectivity will advance the development of novel hypotheses about seizure networks.
Assuntos
Imagem de Difusão por Ressonância Magnética , Epilepsia do Lobo Temporal/patologia , Epilepsia do Lobo Temporal/fisiopatologia , Hipocampo/patologia , Rede Nervosa/patologia , Adulto , Idoso , Lobectomia Temporal Anterior , Imagem de Tensor de Difusão , Epilepsia do Lobo Temporal/diagnóstico por imagem , Epilepsia do Lobo Temporal/cirurgia , Feminino , Hipocampo/diagnóstico por imagem , Humanos , Pessoa de Meia-Idade , Rede Nervosa/diagnóstico por imagem , Adulto JovemRESUMO
Mesoscale diffusion magnetic resonance imaging (MRI) endeavors to bridge the gap between macroscopic white matter tractography and microscopic studies investigating the cytoarchitecture of human brain tissue. To ensure a robust measurement of diffusion at the mesoscale, acquisition parameters were arrayed to investigate their effects on scalar indices (mean, radial, axial diffusivity, and fractional anisotropy) and streamlines (i.e., graphical representation of axonal tracts) in hippocampal layers. A mesoscale resolution afforded segementation of the pyramidal cell layer (CA1-4), the dentate gyrus, as well as stratum moleculare, radiatum, and oriens. Using ex vivo samples, surgically excised from patients with intractable epilepsy (n = 3), we found that shorter diffusion times (23.7 ms) with a b-value of 4,000 s/mm2 were advantageous at the mesoscale, providing a compromise between mean diffusivity and fractional anisotropy measurements. Spatial resolution and sample orientation exerted a major effect on tractography, whereas the number of diffusion gradient encoding directions minimally affected scalar indices and streamline density. A sample temperature of 15°C provided a compromise between increasing signal-to-noise ratio and increasing the diffusion properties of the tissue. Optimization of the acquisition afforded a system's view of intra- and extra-hippocampal connections. Tractography reflected histological boundaries of hippocampal layers. Individual layer connectivity was visualized, as well as streamlines emanating from individual sub-fields. The perforant path, subiculum and angular bundle demonstrated extra-hippocampal connections. Histology of the samples confirmed individual cell layers corresponding to ROIs defined on MR images. We anticipate that this ex vivo mesoscale imaging will yield novel insights into human hippocampal connectivity.
Assuntos
Imagem de Difusão por Ressonância Magnética , Substância Cinzenta/diagnóstico por imagem , Hipocampo/diagnóstico por imagem , Rede Nervosa/diagnóstico por imagem , Via Perfurante/diagnóstico por imagem , Células Piramidais/citologia , Idoso , Lobectomia Temporal Anterior , Giro Denteado/diagnóstico por imagem , Giro Denteado/patologia , Imagem de Difusão por Ressonância Magnética/métodos , Imagem de Difusão por Ressonância Magnética/normas , Imagem de Tensor de Difusão/métodos , Imagem de Tensor de Difusão/normas , Epilepsia do Lobo Temporal/patologia , Epilepsia do Lobo Temporal/cirurgia , Feminino , Substância Cinzenta/patologia , Hipocampo/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Rede Nervosa/patologia , Via Perfurante/patologia , Células Piramidais/patologiaRESUMO
Inhibition of metabolic re-programming represents an attractive approach for prevention of prostate cancer. Studies have implicated increased synthesis of fatty acids or glycolysis in pathogenesis of human prostate cancers. We have shown previously that prostate cancer prevention by sulforaphane (SFN) in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model is associated with inhibition of fatty acid metabolism. This study utilized human prostate cancer cell lines (LNCaP, 22Rv1 and PC-3), two different transgenic mouse models (TRAMP and Hi-Myc) and plasma specimens from a clinical study to explore the glycolysis inhibition potential of SFN. We found that SFN treatment: (i) decreased real-time extracellular acidification rate in LNCaP, but not in PC-3 cell line; (ii) significantly downregulated expression of hexokinase II (HKII), pyruvate kinase M2 and/or lactate dehydrogenase A (LDHA) in vitro in cells and in vivo in neoplastic lesions in the prostate of TRAMP and Hi-Myc mice; and (iii) significantly suppressed glycolysis in prostate of Hi-Myc mice as measured by ex vivo1H magnetic resonance spectroscopy. SFN treatment did not decrease glucose uptake or expression of glucose transporters in cells. Overexpression of c-Myc, but not constitutively active Akt, conferred protection against SFN-mediated downregulation of HKII and LDHA protein expression and suppression of lactate levels. Examination of plasma lactate levels in prostate cancer patients following administration of an SFN-rich broccoli sprout extract failed to show declines in its levels. Additional clinical trials are needed to determine whether SFN treatment can decrease lactate production in human prostate tumors.
Assuntos
Adenocarcinoma/metabolismo , Anticarcinógenos/farmacologia , Glicólise/efeitos dos fármacos , Isotiocianatos/farmacologia , Neoplasias da Próstata/metabolismo , Adenocarcinoma/patologia , Animais , Quimioprevenção/métodos , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neoplasias da Próstata/patologia , SulfóxidosRESUMO
Na+ /H+ exchanger (NHE1) activation is required for multiple microglial functions. We investigated effects of selective deletion of microglial Nhe1 in Cx3cr1-CreER ;Nhe1f/f mice on neuroinflammation and tissue repair after ischemic stroke. Infarct volume was similar in corn oil or tamoxifen (Tam)-treated mice at 48 hr and 14 days post-stroke. However, the Tam-treated mice showed significantly higher survival rate and faster neurological function recovery during day 1-14 post-stroke. Deletion of microglial Nhe1 prevented the elevation of CD11b+ /CD45low-med microglia in the ischemic hemisphere at day 3 post-stroke, but stimulated expression of Ym1, CD68, TGF-ß, IL-10, decreased expression of CD86 and IL-1ß, and reduced GFAP+ reactive astrocytes. Moreover, at day 14 post-stroke, enhanced white matter myelination was detected in the microglial Nhe1 deleted mice. In comparison, neuronal Nhe1-null mice (the CamKII-Cre+/- ;Nhe1f/f mice) showed a significant reduction in both acute and subacute infarct volume, along with increased survival rate and moderate neurological function recovery. However, these neuronal Nhe1-null mice did not exhibit reduced activation of CD11b+ /CD45low-med microglia or CD11b+ /CD45hi macrophages in the ischemic brains, and they exhibited no reductions in white matter lesions. Taken together, this study demonstrated that deletion of microglial and neuronal Nhe1 had differential effects on ischemic brain damage. Microglial NHE1 is involved in pro-inflammatory responses during post-stroke brain tissue repair. In contrast, neuronal NHE1 activation is directly associated with the acute ischemic neuronal injury but not inflammation. Our study reveals that NHE1 protein is a potential therapeutic target critical for differential regulation of ischemic neuronal injury, demyelination and tissue repair.
Assuntos
Receptor 1 de Quimiocina CX3C/metabolismo , Doenças Desmielinizantes/etiologia , Infarto da Artéria Cerebral Média/complicações , Microglia/metabolismo , Recuperação de Função Fisiológica/fisiologia , Trocador 1 de Sódio-Hidrogênio/metabolismo , Substância Branca/patologia , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Receptor 1 de Quimiocina CX3C/genética , Proteínas de Ligação ao Cálcio/metabolismo , Doenças Desmielinizantes/tratamento farmacológico , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Infarto da Artéria Cerebral Média/diagnóstico por imagem , Infarto da Artéria Cerebral Média/tratamento farmacológico , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Recuperação de Função Fisiológica/efeitos dos fármacos , Trocador 1 de Sódio-Hidrogênio/genética , Distúrbios Somatossensoriais/etiologia , Tamoxifeno/farmacologia , Substância Branca/diagnóstico por imagemRESUMO
Here, we tested whether combined contrast-enhanced magnetic resonance imaging (CCE-MRI), using a mixture of gadolinium- and iron oxide-based contrast agents, can segment the bladder wall from the bladder lumen. CCE-MRI relies on the differences in particle size and contrast mechanisms of two agents for improved image contrast. Under isoflurane anesthesia, T1-weighted imaging of adult female Sprague-Dawley rat bladder was performed using standard turbospin echo sequences at 7 Tesla, before and after transurethral instillation of 0.3 ml of single-contrast MRI or CCE-MRI composed of 0.4-64 mM of gadolinium chelate (Gd-DTPA/Gadavist) and 5 mM ferumoxytol. Bladder wall contrast was assessed in the control group exposed to saline and in the bladder injury group exposed to 0.5 ml of protamine sulfate (10 mg/ml) for 30 min. CCE-MRI following instillation of 0.4-4 mM Gd-DTPA and 5 mM ferumoxytol mixture achieved segmentation between the bladder lumen and bladder wall. Hyperintensity in the bladder wall combined with hypointensity in the lumen is consistent with the increased diffusion of the dissolved Gd-DTPA and simultaneous localization of the larger nanoparticles of ferumoxytol in the lumen. The normalized hyperintense signal in the bladder wall increased from 0.46 ± 0.07 in control group to 0.73 ± 0.14 in the protamine sulfate-exposed group (P < 0.0001). CCE-MRI following instillation of contrast mixture identifies bladder wall changes likely associated with bladder injury with improved image contrast.
Assuntos
Meios de Contraste/administração & dosagem , Óxido Ferroso-Férrico/administração & dosagem , Imageamento por Ressonância Magnética/métodos , Compostos Organometálicos/administração & dosagem , Doenças da Bexiga Urinária/diagnóstico por imagem , Bexiga Urinária/diagnóstico por imagem , Administração Intravesical , Animais , Modelos Animais de Doenças , Feminino , Nanopartículas , Tamanho da Partícula , Valor Preditivo dos Testes , Protaminas , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Fatores de Tempo , Bexiga Urinária/patologia , Doenças da Bexiga Urinária/induzido quimicamente , Doenças da Bexiga Urinária/patologiaRESUMO
BACKGROUND: Disturbances in cerebral blood flow (CBF) and brain oxygenation (PbO2) are present early after pediatric cardiac arrest (CA). CBF-targeted therapies improved neurological outcome in our CA model. To assess the therapeutic window for CBF- and PbO2-targeted therapies, we propose to determine if CBF and PbO2 disturbances persist at 24 h after experimental pediatric CA. METHODS: Regional CBF and PbO2 were measured at 24 h after asphyxial CA in immature rats (n = 26, 6-8/group) using arterial spin label MRI and tissue electrodes, respectively. RESULTS: In all regions but the thalamus, CBF recovered to sham values by 24 h; thalamic CBF was >32% higher after CA vs. sham. PbO2 values at 24 h after CA in the cortex and thalamus were similar to shams in rats who received supplemental oxygen, however, on room air, cortical PbO2 was lower after CA vs. shams. CONCLUSION: CBF remains increased in the thalamus at 24 h after CA and PbO2 is decreased to hypoxic levels in cortex at 24 h after CA in rats who do not receive supplemental oxygen. Given the enduring disturbances in this model and the lack of routine CBF or PbO2 monitoring in patients, our data suggest the need for clinical correlation.
Assuntos
Asfixia/fisiopatologia , Encéfalo/fisiopatologia , Circulação Cerebrovascular/fisiologia , Parada Cardíaca/fisiopatologia , Consumo de Oxigênio/fisiologia , Animais , Asfixia/terapia , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Parada Cardíaca/terapia , Hipóxia/fisiopatologia , Hipóxia/terapia , Masculino , Oxigênio/uso terapêutico , Ratos , Ratos Sprague-Dawley , Tálamo/irrigação sanguínea , Tálamo/metabolismoRESUMO
PURPOSE: The ability to detect the migration of cells in living organisms is fundamental in understanding biological processes and important for the development of novel cell-based therapies to treat disease. MRI can be used to detect the migration of cells labeled with superparamagnetic iron-oxide (SPIO) or perfluorocarbon (PFC) agents. In this study, we explored combining these two cell-labeling approaches to overcome current limitations and enable new applications for cellular MRI. METHODS: We characterized (19)F-NMR relaxation properties of PFC-labeled cells in the presence of SPIO and imaged cells both ex vivo and in vivo in a rodent inflammation model to demonstrate selective visualization of cell populations. RESULTS: We show that with UTE3D, RARE, and FLASH (19) F images one can uniquely identify PFC-labeled cells, colocalized PFC- and SPIO-labeled cells, and PFC/SPIO-colabeled cells. CONCLUSION: This new methodology has the ability to improve and expand applications of MRI cell tracking. Combining PFC and SPIO strategies can potentially provide a method to quench PFC signal transferred from dead cells to macrophages, thereby eliminating false positives. In addition, combining these techniques could also be used to track two cell types simultaneously and probe cell-cell proximity in vivo with MRI.
Assuntos
Rastreamento de Células/métodos , Dextranos , Fluorocarbonos , Aumento da Imagem/métodos , Macrófagos/patologia , Imageamento por Ressonância Magnética/métodos , Nanopartículas de Magnetita , Animais , Meios de Contraste , Estudos de Viabilidade , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Coloração e Rotulagem/métodosRESUMO
OBJECT Craniosynostosis is a condition in which one or more of the calvarial sutures fuses prematurely. In addition to the cosmetic ramifications attributable to premature suture fusion, aberrations in neurophysiological parameters are seen, which may result in more significant damage. This work examines the microstructural integrity of white matter, using diffusion tensor imaging (DTI) in a homogeneous strain of rabbits with simple, familial coronal suture synostosis before and after surgical correction. METHODS After diagnosis, rabbits were assigned to different groups: wild-type (WT), rabbits with early-onset complete fusion of the coronal suture (BC), and rabbits that had undergone surgical correction with suturectomy (BC-SU) at 10 days of age. Fixed rabbit heads were imaged at 12, 25, or 42 days of life using a 4.7-T, 40-cm bore Avance scanner with a 7.2-cm radiofrequency coil. For DTI, a 3D spin echo sequence was used with a diffusion gradient (b = 2000 sec/mm(2)) applied in 6 directions. RESULTS As age increased from 12 to 42 days, the DTI differences between WT and BC groups became more pronounced (p < 0.05, 1-way ANOVA), especially in the corpus callosum, cingulum, and fimbriae. Suturectomy resulted in rabbits with no significant differences compared with WT animals, as assessed by DTI of white matter tracts. Also, it was possible to predict to which group an animal belonged (WT, BC, and BC-SU) with high accuracy based on imaging data alone using a linear support vector machine classifier. The ability to predict to which group the animal belonged improved as the age of the animal increased (71% accurate at 12 days and 100% accurate at 42 days). CONCLUSIONS Craniosynostosis results in characteristic changes of major white matter tracts, with differences becoming more apparent as the age of the rabbits increases. Early suturectomy (at 10 days of life) appears to mitigate these differences.
Assuntos
Craniossinostoses/patologia , Craniossinostoses/cirurgia , Substância Branca/patologia , Substância Branca/cirurgia , Animais , Craniossinostoses/metabolismo , Imagem de Tensor de Difusão/métodos , Coelhos , Substância Branca/metabolismoRESUMO
BACKGROUND: Superparamagnetic iron-oxide nanoparticles are useful as contrast agents for anatomical, functional and cellular MRI, drug delivery agents, and diagnostic biosensors. Nanoparticles are generally cleared by the reticuloendothelial system (RES), in particular taken up by Kupffer cells in the liver, limiting particle bioavailability and in-vivo applications. Strategies that decrease the RES clearance and prolong the circulation residence time of particles can improve the in-vivo targeting efficiency. METHODS: Intralipid 20.0%, an FDA approved nutritional supplement, was intravenously administered in rats at the clinical dose (2g/kg) 1h before intravenous injection of ultra-small superparamagnetic iron-oxide (USPIO) or micron-sized paramagnetic iron-oxide (MPIO) particles. Blood half-life, monocyte labeling efficiency, and particle biodistribution were assessed by magnetic resonance relaxometry, flow cytometry, inductively-coupled plasma MS, and histology. RESULTS: Pre-treatment with Intralipid resulted in a 3.1-fold increase in USPIO blood half-life and a 2-fold increase in USPIO-labeled monocytes. A 2.5-fold increase in MPIO blood half-life and a 5-fold increase in MPIO-labeled monocytes were observed following Intralipid pre-treatment, with a 3.2-fold increase in mean iron content up to 2.60pg Fe/monocyte. With Intralipid, there was a 49.2% and 45.1% reduction in liver uptake vs. untreated controls at 48h for USPIO and MPIO, respectively. CONCLUSIONS: Intralipid pre-treatment significantly decreases initial RES uptake and increases in-vivo circulation and blood monocyte labeling efficiency for nano- and micron-sized superparamagnetic iron-oxide particles. GENERAL SIGNIFICANCE: Our findings can have broad applications for imaging and drug delivery applications, increasing the bioavailability of nano- and micron-sized particles for target sites other than the liver.
Assuntos
Meios de Contraste/farmacocinética , Emulsões Gordurosas Intravenosas/farmacocinética , Compostos Férricos/farmacocinética , Células de Kupffer/metabolismo , Fígado/metabolismo , Nanopartículas de Magnetita , Fosfolipídeos/farmacocinética , Óleo de Soja/farmacocinética , Animais , Disponibilidade Biológica , Meios de Contraste/farmacologia , Emulsões/farmacocinética , Emulsões/farmacologia , Emulsões Gordurosas Intravenosas/farmacologia , Compostos Férricos/farmacologia , Meia-Vida , Células de Kupffer/citologia , Fígado/citologia , Masculino , Fosfolipídeos/farmacologia , Ratos , Ratos Endogâmicos BN , Óleo de Soja/farmacologiaRESUMO
BACKGROUND: Liver cancer is one of the most lethal malignancies for humans. The treatment options for advanced-stage liver cancer remain limited. A new treatment is urgently needed to reduce the mortality of the disease. METHODS: In this report, we developed a technology for mutation site insertion of a suicide gene (herpes simplex virus type 1- thymidine kinase) based on type II CRISPR RNA-guided endonuclease Cas9-mediated genome editing to treat liver cancers. RESULTS: We applied the strategy to 3 different mutations: S45P mutation of catenin beta 1, chromosome breakpoint of solute carrier family 45 member 2-alpha-methylacyl-CoA racemase gene fusion, and V235G mutation of SAFB-like transcription modulator. The results showed that the herpes simplex virus type 1-thymidine kinase insertion rate at the S45P mutation site of catenin beta 1 reached 77.8%, while the insertion rates at the breakpoint of solute carrier family 45 member 2 - alpha-methylacyl-CoA racemase gene fusion were 95.1%-98.7%, and the insertion at V235G of SAFB-like transcription modulator was 51.4%. When these targeting reagents were applied to treat mouse spontaneous liver cancer induced by catenin beta 1S45P or solute carrier family 45 member 2-alpha-methylacyl-CoA racemase, the mice experienced reduced tumor burden and increased survival rate. Similar results were also obtained for the xenografted liver cancer model: Significant reduction of tumor volume, reduction of metastasis rate, and improved survival were found in mice treated with the targeting reagent, in comparison with the control-treated groups. CONCLUSIONS: Our studies suggested that mutation targeting may hold promise as a versatile and effective approach to treating liver cancers.