Diphtheria toxin induced but not CSF1R inhibitor mediated microglia ablation model leads to the loss of CSF/ventricular spaces in vivo that is independent of cytokine upregulation.

BACKGROUND: Two recently developed novel rodent models have been reported to ablate microglia, either by genetically targeting microglia (via Cx3cr1-creER: iDTR + Dtx) or through pharmacologically targeting the CSF1R receptor with its inhibitor (PLX5622). Both models have been widely used in recent years to define essential functions of microglia and have led to high impact studies that have moved the field forward. METHODS: Using either Cx3cr1-iDTR mice in combination with Dtx or via the PLX5622 diet to pharmacologically ablate microglia, we compared the two models via MRI and histology to study the general anatomy of the brain and the CSF/ventricular systems. Additionally, we analyzed the cytokine profile in both microglia ablation models. RESULTS: We discovered that the genetic ablation (Cx3cr1-iDTR + Dtx), but not the pharmacological microglia ablation (PLX5622), displays a surprisingly rapid pathological condition in the brain represented by loss of CSF/ventricles without brain parenchymal swelling. This phenotype was observed both in MRI and histological analysis. To our surprise, we discovered that the iDTR allele alone leads to the loss of CSF/ventricles phenotype following diphtheria toxin (Dtx) treatment independent of cre expression. To examine the underlying mechanism for the loss of CSF in the Cx3cr1-iDTR ablation and iDTR models, we additionally investigated the cytokine profile in the Cx3cr1-iDTR + Dtx, iDTR + Dtx and the PLX models. We found increases of multiple cytokines in the Cx3cr1-iDTR + Dtx but not in the pharmacological ablation model nor the iDTR + Dtx mouse brains at the time of CSF loss (3 days after the first Dtx injection). This result suggests that the upregulation of cytokines is not the cause of the loss of CSF, which is supported by our data indicating that brain parenchyma swelling, or edema are not observed in the Cx3cr1-iDTR + Dtx microglia ablation model. Additionally, pharmacological inhibition of the KC/CXCR2 pathway (the most upregulated cytokine in the Cx3cr1-iDTR + Dtx model) did not resolve the CSF/ventricular loss phenotype in the genetic microglia ablation model. Instead, both the Cx3cr1-iDTR + Dtx ablation and iDTR + Dtx models showed increased activated IBA1 + cells in the choroid plexus (CP), suggesting that CP-related pathology might be the contributing factor for the observed CSF/ventricular shrinkage phenotype. CONCLUSIONS: Our data, for the first time, reveal a robust and global CSF/ventricular space shrinkage pathology in the Cx3cr1-iDTR genetic ablation model caused by iDTR allele, but not in the PLX5622 ablation model, and suggest that this pathology is not due to brain edema formation but to CP related pathology. Given the wide utilization of the iDTR allele and the Cx3cr1-iDTR model, it is crucial to fully characterize this pathology to understand the underlying causal mechanisms. Specifically, caution is needed when utilizing this model to interpret subtle neurologic functional changes that are thought to be mediated by microglia but could, instead, be due to CSF/ventricular loss in the genetic ablation model.

MRI Measures of Murine Liver Fibrosis.

BACKGROUND: An imaging method that allows quantitative fibrosis estimates is needed to facilitate the diagnosis of chronic liver disease. Amide proton transfer (APT) and tissue sodium concentration (TSC) estimates could meet this need. HYPOTHESIS: APT and TSC estimates correlate with fibrosis in a mouse model of chronic liver disease. STUDY TYPE: Prospective. PHANTOMS/ANIMAL MODEL: Male C57Bl/6 mice given CCl4 or vehicle (N = 8 each) twice weekly for 16 weeks. FIELD STRENGTH/SEQUENCE: Liver T1 (Look-Locker gradient recalled echo [GRE] sequence), T2 (multiecho spin echo sequence), T1rho (fast spin echo sequence with 500 Hz spin locking pulse), and APT (GRE sequence with off-resonance pulses) data were acquired at 7 T at 12 and 16 weeks. Liver sodium data (multiple echo GRE sequence) were acquired at 12 weeks at 9.4 T. ASSESSMENT: Liver proton T1 , T2 , T1rho , APT, sodium T2 *, and TSC were calculated. Histological measures included Sirius Red, hematoxylin and eosin, liver hydroxyproline content, and serum alanine transaminase (ALT). STATISTICAL TESTS: Welch's two-sided t-test was used to test for differences between control and CCl4 -treated groups for serum ALT, hydroxyproline, Sirius Red staining, T1 , T2 , T1rho , APT, TSC, and sodium T2 *. Pearson's correlations between liver T1 , APT, TSC, or sodium T2 * with Sirius Red staining and hydroxyproline levels were calculated. RESULTS: APT was significantly different (P < 0.05) between groups in the left liver lobe at 16 weeks (CCl4 : 8.0% ± 1.2%, controls: 6.2% ± 1.0%), as were average liver TSC at 12 weeks (CCl4 : 38 mM ± 5 mM, controls: 27 mM ± 2 mM), and average sodium liver T2 * at 12 weeks (CCl4 : 10 msec ± 1.0 msec, controls: 12 msec ± 1.9 msec). APT, TSC, and sodium T2 * correlated significantly (P < 0.05) with Sirius Red staining and hydroxyproline levels. DATA CONCLUSION: Liver TSC and APT significantly correlated with histopathologic markers of fibrosis in this mouse model. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 3.

Ablation of polyamine catabolic enzymes provokes Purkinje cell damage, neuroinflammation, and severe ataxia.

BACKGROUND: Polyamine catabolism plays a key role in maintaining intracellular polyamine pools, yet its physiological significance is largely unexplored. Here, we report that the disruption of polyamine catabolism leads to severe cerebellar damage and ataxia, demonstrating the fundamental role of polyamine catabolism in the maintenance of cerebellar function and integrity. METHODS: Mice with simultaneous deletion of the two principal polyamine catabolic enzymes, spermine oxidase and spermidine/spermine N1-acetyltransferase (Smox/Sat1-dKO), were generated by the crossbreeding of Smox-KO (Smox-/-) and Sat1-KO (Sat1-/-) animals. Development and progression of tissue injury was monitored using imaging, behavioral, and molecular analyses. RESULTS: Smox/Sat1-dKO mice are normal at birth, but develop progressive cerebellar damage and ataxia. The cerebellar injury in Smox/Sat1-dKO mice is associated with Purkinje cell loss and gliosis, leading to neuroinflammation and white matter demyelination during the latter stages of the injury. The onset of tissue damage in Smox/Sat1-dKO mice is not solely dependent on changes in polyamine levels as cerebellar injury was highly selective. RNA-seq analysis and confirmatory studies revealed clear decreases in the expression of Purkinje cell-associated proteins and significant increases in the expression of transglutaminases and markers of neurodegenerative microgliosis and astrocytosis. Further, the α-Synuclein expression, aggregation, and polyamination levels were significantly increased in the cerebellum of Smox/Sat1-dKO mice. Finally, there were clear roles of transglutaminase-2 (TGM2) in the cerebellar pathologies manifest in Smox/Sat1-dKO mice, as pharmacological inhibition of transglutaminases reduced the severity of ataxia and cerebellar injury in Smox/Sat1-dKO mice. CONCLUSIONS: These results indicate that the disruption of polyamine catabolism, via coordinated alterations in tissue polyamine levels, elevated transglutaminase activity and increased expression, polyamination, and aggregation of α-Synuclein, leads to severe cerebellar damage and ataxia. These studies indicate that polyamine catabolism is necessary to Purkinje cell survival, and for sustaining the functional integrity of the cerebellum.

Preclinical hyperpolarized 129 Xe MRI: ventilation and T2 * mapping in mouse lungs at 7 T using multi-echo flyback UTE.

Fast apparent transverse relaxation (short T2 *) is a common obstacle when attempting to perform quantitative 1 H MRI of the lungs. While T2 * times are longer for pulmonary hyperpolarized (HP) gas functional imaging (in particular for gaseous 129 Xe), T2 * can still lead to quantitative inaccuracies for sequences requiring longer echo times (such as diffusion weighted images) or longer readout duration (such as spiral sequences). This is especially true in preclinical studies, where high magnetic fields lead to shorter relaxation times than are typically seen in human studies. However, the T2 * of HP 129 Xe in the most common animal model of human disease (mice) has not been reported. Herein, we present a multi-echo radial flyback imaging sequence and use it to measure HP 129 Xe T2 * at 7 T under a variety of respiratory conditions. This sequence mitigates the impact of T1 relaxation outside the animal by using multiple gradient-refocused echoes to acquire images at a number of effective echo times for each RF excitation. After validating the sequence using a phantom containing water doped with superparamagnetic iron oxide nanoparticles, we measured the 129 Xe T2 * in vivo for 10 healthy C57Bl/6 J mice and found T2 * ~ 5 ms in the lung airspaces. Interestingly, T2 * was relatively constant over all experimental conditions, and varied significantly with sex, but not age, mass, or the O2 content of the inhaled gas mixture. These results are discussed in the context of T2 * relaxation within porous media.

Loss of SLC25A46 causes neurodegeneration by affecting mitochondrial dynamics and energy production in mice.

Recently, we identified biallelic mutations of SLC25A46 in patients with multiple neuropathies. Functional studies revealed that SLC25A46 may play an important role in mitochondrial dynamics by mediating mitochondrial fission. However, the cellular basis and pathogenic mechanism of the SLC25A46-related neuropathies are not fully understood. Thus, we generated a Slc25a46 knock-out mouse model. Mice lacking SLC25A46 displayed severe ataxia, mainly caused by degeneration of Purkinje cells. Increased numbers of small, unmyelinated and degenerated optic nerves as well as loss of retinal ganglion cells indicated optic atrophy. Compound muscle action potentials in peripheral nerves showed peripheral neuropathy associated with degeneration and demyelination in axons. Mutant cerebellar neurons have large mitochondria, which exhibit abnormal distribution and transport. Biochemically mutant mice showed impaired electron transport chain activity and accumulated autophagy markers. Our results suggest that loss of SLC25A46 causes degeneration in neurons by affecting mitochondrial dynamics and energy production.

Omega-3 fatty acid deficiency impairs frontostriatal recruitment following repeated amphetamine treatment in rats: A 7 Tesla in vivo phMRI study.

Although attention deficit hyperactivity disorder is associated with deficits in docosahexaenoic acid (DHA), an omega-3 fatty acid implicated in dopamine and glutamate synaptic plasticity, its role in neuroplastic brain changes that occur following repeated amphetamine (AMPH) treatment are not known. This study used pharmacological magnetic resonance imaging to investigate the impact of repeated AMPH exposure and alterations in brain DHA levels on AMPH-induced brain activation patterns. Male rats were fed a diet with no n-3 fatty acids (Deficient, DEF, n = 20), a diet fortified with preformed DHA (fish oil, FO, n = 20), or a control diet fortified with alpha-linolenic acid (n = 20) from P21 to P90. During adolescence (P40-60), one-half of each diet group received daily AMPH injections escalated weekly (0.5, 1.0, 2.5, 5.0 mg/kg/d) or drug vehicle. Following a 30-d abstinence period blood oxygen level dependent (BOLD) responses were determined in a 7 T Bruker Biospec system following an AMPH challenge (7.5 mg/kg, i.v). Postmortem erythrocyte and forebrain DHA composition were determined by gas chromatography. Compared with control rats, forebrain and erythrocyte DHA levels were significantly lower in DEF rats and significantly higher in FO rats. Across AMPH doses DEF rats exhibited greater locomotor activity compared to control and FO rats. In AMPH-naïve rats, the AMPH challenge increased BOLD activity in the substantia nigra and basal forebrain and no diet group differences were observed. In AMPH-pretreated control and FO rats, the AMPH challenge similarly increased BOLD activation in the bilateral caudate putamen, thalamus, and motor and cingulate cortices. In contrast, BOLD activation in AMPH-pretreated DEF rats was similar to AMPH-naïve DEF animals, and AMPH-pretreated DEF rats exhibited attenuated frontostriatal BOLD activation compared with AMPH-pretreated control and FO rats. These findings demonstrate that chronic escalating AMPH treatment induces enduring frontostriatal recruitment and that peri-adolescent deficits in brain DHA accrual impair this response.

Deficits in Docosahexaenoic Acid Accrual during Adolescence Reduce Rat Forebrain White Matter Microstructural Integrity: An in vivo Diffusion Tensor Imaging Study.

Neuropsychiatric disorders that frequently initially emerge during adolescence are associated with deficits in the omega-3 (n-3) fatty acid docosahexaenoic acid (DHA), elevated proinflammatory signaling, and regional reductions in white matter integrity (WMI). This study determined the effects of altering brain DHA accrual during adolescence on WMI in the rat brain by diffusion tensor imaging (DTI), and investigated the potential mediating role of proinflammatory signaling. During periadolescent development, male rats were fed a diet deficient in n-3 fatty acids (DEF, n = 20), a fish oil-fortified diet containing preformed DHA (FO, n = 20), or a control diet (CON, n = 20). In adulthood, DTI scans were performed and brain WMI was determined using voxelwise tract-based spatial statistics (TBSS). Postmortem fatty acid composition, peripheral (plasma IL-1ß, IL-6, and C-reactive protein [CRP]) and central (IL-1ß and CD11b mRNA) proinflammatory markers, and myelin basic protein (MBP) mRNA expression were determined. Compared with CON rats, forebrain DHA levels were lower in DEF rats and higher in FO rats. Compared with CON rats, DEF rats exhibited greater radial diffusivity (RD) and mean diffusivity in the right external capsule, and greater axial diffusivity in the corpus callosum genu and left external capsule. DEF rats also exhibited greater RD than FO rats in the right external capsule. Forebrain MBP expression did not differ between groups. Compared with CON rats, central (IL-1ß and CD11b) and peripheral (IL-1ß and IL-6) proinflammatory markers were not different in DEF rats, and DEF rats exhibited lower CRP levels. These findings demonstrate that deficits in adolescent DHA accrual negatively impact forebrain WMI, independently of elevated proinflammatory signaling.

Intravenous miR-144 inhibits tumor growth in diethylnitrosamine-induced hepatocellular carcinoma in mice.

Previous in vitro studies have demonstrated that miR-144 inhibits hepatocellular carcinoma cell proliferation, invasion, and migration. We have shown that miR-144, injected intravenously, is taken up by the liver and induces endogenous hepatic synthesis of miR-144. We hypothesized that administered miR-144 has tumor-suppressive effects on liver tumor development in vivo. The effects of miR-144 on tumorigenesis and tumor growth were tested in a diethylnitrosamine-induced hepatocellular carcinoma mouse model. MiR-144 injection had no effect on body weight but significantly reduced diethylnitrosamine-induced liver enlargement compared with scrambled microRNA. MiR-144 had no effect on diethylnitrosamine-induced liver tumor number but reduced the tumor size above 50%, as evaluated by magnetic resonance imaging (scrambled microRNA 23.07 ± 5.67 vs miR-144 10.38 ± 2.62, p < 0.05) and histological analysis (scrambled microRNA 30.75 ± 5.41 vs miR-144 15.20 ± 3.41, p < 0.05). The levels of miR-144 was suppressed in tumor tissue compared with non-tumor tissue in all treatment groups (diethylnitrosamine-phosphate-buffered saline non-tumor 1.05 ± 0.09 vs tumor 0.54 ± 0.08, p < 0.01; diethylnitrosamine-scrambled microRNA non-tumor 1.23 ± 0.33 vs tumor 0.44 ± 0.10, p < 0.05; diethylnitrosamine-miR-144 non-tumor 54.72 ± 11.80 vs tumor 11.66 ± 2.75, p < 0.01), but injection of miR-144 greatly increased miR-144 levels both in tumor and non-tumor tissues. Mechanistic studies showed that miR-144 targets epidermal growth factor receptor and inhibits the downstream Src/AKT signaling pathway which has previously been implicated in hepatocellular carcinoma tumorigenesis. Exogenously delivered miR-144 may be a therapeutic strategy to suppress tumor growth in hepatocellular carcinoma.

Blocking lymphocyte trafficking with FTY720 prevents inflammation-sensitized hypoxic-ischemic brain injury in newborns.

Intrauterine infection (chorioamnionitis) aggravates neonatal hypoxic-ischemic (HI) brain injury, but the mechanisms linking systemic inflammation to the CNS damage remain uncertain. Here we report evidence for brain influx of T-helper 17 (TH17)-like lymphocytes to coordinate neuroinflammatory responses in lipopolysaccharide (LPS)-sensitized HI injury in neonates. We found that both infants with histological chorioamnionitis and rat pups challenged by LPS/HI have elevated expression of the interleukin-23 (IL-23) receptor, a marker of early TH17 lymphocytes, in the peripheral blood mononuclear cells. Post-LPS/HI administration of FTY720 (fingolimod), a sphingosine-1-phosphate receptor agonist that blocks lymphocyte trafficking, mitigated the influx of leukocytes through the choroid plexus and acute induction of nuclear factor-κB signaling in the brain. Subsequently, the FTY720 treatment led to attenuated blood-brain barrier damage, fewer cluster of differentiation 4-positive, IL-17A-positive T-cells in the brain, less proinflammatory cytokine, and better preservation of growth and white matter functions. The FTY720 treatment also provided dose-dependent reduction of brain atrophy, rescuing >90% of LPS/HI-induced brain tissue loss. Interestingly, FTY720 neither opposed pure-HI brain injury nor directly inhibited microglia in both in vivo and in vitro models, highlighting its unique mechanism against inflammation-sensitized HI injury. Together, these results suggest that the dual hit of systemic inflammation and neonatal HI injury triggers early onset of the TH17/IL-17-mediated immunity, which causes severe brain destruction but responds remarkably to the therapeutic blockade of lymphocyte trafficking.

Plasminogen activator inhibitor-1 mitigates brain injury in a rat model of infection-sensitized neonatal hypoxia-ischemia.

Intrauterine infection exacerbates neonatal hypoxic-ischemic (HI) brain injury and impairs the development of cerebral cortex. Here we used low-dose lipopolysaccharide (LPS) pre-exposure followed by unilateral cerebral HI insult in 7-day-old rats to study the pathogenic mechanisms. We found that LPS pre-exposure blocked the HI-induced proteolytic activity of tissue-type plasminogen activator (tPA), but significantly enhanced NF-κB signaling, microglia activation, and the production of pro-inflammatory cytokines in newborn brains. Remarkably, these pathogenic responses were all blocked by intracerebroventricular injection of a stable-mutant form of plasminogen activator protein-1 called CPAI. Similarly, LPS pre-exposure amplified, while CPAI therapy mitigated HI-induced blood-brain-barrier damage and the brain tissue loss with a therapeutic window at 4 h after the LPS/HI insult. The CPAI also blocks microglia activation following a brain injection of LPS, which requires the contribution by tPA, but not the urinary-type plasminogen activator (uPA), as shown by experiments in tPA-null and uPA-null mice. These results implicate the nonproteolytic tPA activity in LPS/HI-induced brain damage and microglia activation. Finally, the CPAI treatment protects near-normal motor and white matter development despite neonatal LPS/HI insult. Together, because CPAI blocks both proteolytic and nonproteolytic tPA neurotoxicity, it is a promising therapeutics of neonatal HI injury either with or without infection.

Single-cell sequencing dissects the transcriptional identity of activated fibroblasts and identifies novel persistent distal tubular injury patterns in kidney fibrosis.

Examining kidney fibrosis is crucial for mechanistic understanding and developing targeted strategies against chronic kidney disease (CKD). Persistent fibroblast activation and tubular epithelial cell (TEC) injury are key CKD contributors. However, cellular and transcriptional landscapes of CKD and specific activated kidney fibroblast clusters remain elusive. Here, we analyzed single cell transcriptomic profiles of two clinically relevant kidney fibrosis models which induced robust kidney parenchymal remodeling. We dissected the molecular and cellular landscapes of kidney stroma and newly identified three distinctive fibroblast clusters with "secretory", "contractile" and "vascular" transcriptional enrichments. Also, both injuries generated failed repair TECs (frTECs) characterized by decline of mature epithelial markers and elevation of stromal and injury markers. Notably, frTECs shared transcriptional identity with distal nephron segments of the embryonic kidney. Moreover, we identified that both models exhibited robust and previously unrecognized distal spatial pattern of TEC injury, outlined by persistent elevation of renal TEC injury markers including Krt8 and Vcam1, while the surviving proximal tubules (PTs) showed restored transcriptional signature. We also found that long-term kidney injuries activated a prominent nephrogenic signature, including Sox4 and Hox gene elevation, which prevailed in the distal tubular segments. Our findings might advance understanding of and targeted intervention in fibrotic kidney disease.

Lithium compartmentation in brain by 7Li MRS: effect of total lithium concentration.

In previous work at 4.7 T, the individual components of biexponential (7) Li transverse (T2 ) spin relaxation in rat brain in vivo were tentatively identified with intra- and extracellular Li. The goal in this work was to estimate Li's compartmental distribution as a function of total Li concentration in brain from the biexponential decays. Here a localized, biexponential (7) Li T2 MR spin-relaxation study with isotopically enriched (7) LiCl is reported in rat brain in vivo at 7 T. Additionally, a simple linear interpolation using the biexponential T2 values to estimate intracellular Li from individual monoexponential T2 decays was assessed. Intracellular T2 was 14.8 ± 4.3 ms and extracellular T2 was 295 ± 61 ms. The fraction of intracellular brain Li ranged from 37.3 to 64.8% (mean 54.5 ± 6.7%) and did not correlate with total Li concentration. The estimated intracellular Li concentration ranged from 47 to 80% (mean 68.3 ± 8.5%) of the total brain Li concentration and was highly correlated with it. The monoexponential estimates of the intracellular-Li fractions and derived concentrations averaged about 15% higher than the corresponding biexponential estimates. This work supports the previous conclusion that a large fraction of Li in the brain is within the intracellular compartment.

Early postnatal microglial ablation in the Ccdc39 mouse model reveals adverse effects on brain development and in neonatal hydrocephalus.

BACKGROUND: Neonatal hydrocephalus is a congenital abnormality resulting in an inflammatory response and microglial cell activation both clinically and in animal models. Previously, we reported a mutation in a motile cilia gene, Ccdc39 that develops neonatal progressive hydrocephalus (prh) with inflammatory microglia. We discovered significantly increased amoeboid-shaped activated microglia in periventricular white matter edema, reduced mature homeostatic microglia in grey matter, and reduced myelination in the prh model. Recently, the role of microglia in animal models of adult brain disorders was examined using cell type-specific ablation by colony-stimulating factor-1 receptor (CSF1R) inhibitor, however, little information exists regarding the role of microglia in neonatal brain disorders such as hydrocephalus. Therefore, we aim to see if ablating pro-inflammatory microglia, and thus suppressing the inflammatory response, in a neonatal hydrocephalic mouse line could have beneficial effects. METHODS: In this study, Plexxikon 5622 (PLX5622), a CSF1R inhibitor, was subcutaneously administered to wild-type (WT) and prh mutant mice daily from postnatal day (P) 3 to P7. MRI-estimated brain volume was compared with untreated WT and prh mutants P7-9 and immunohistochemistry of the brain sections was performed at P8 and P18-21. RESULTS: PLX5622 injections successfully ablated IBA1-positive microglia in both the WT and prh mutants at P8. Of the microglia that are resistant to PLX5622 treatment, there was a higher percentage of amoeboid-shaped microglia, identified by morphology with retracted processes. In PLX-treated prh mutants, there was increased ventriculomegaly and no change in the total brain volume was observed. Also, the PLX5622 treatment significantly reduced myelination in WT mice at P8, although this was recovered after full microglia repopulation by P20. Microglia repopulation in the mutants worsened hypomyelination at P20. CONCLUSIONS: Microglia ablation in the neonatal hydrocephalic brain does not improve white matter edema, and actually worsens ventricular enlargement and hypomyelination, suggesting critical functions of homeostatic ramified microglia to better improve brain development with neonatal hydrocephalus. Future studies with detailed examination of microglial development and status may provide a clarification of the need for microglia in neonatal brain development.

Single-cell sequencing dissects the transcriptional identity of activated fibroblasts and identifies novel persistent distal tubular injury patterns in kidney fibrosis.

Examining kidney fibrosis is crucial for mechanistic understanding and developing targeted strategies against chronic kidney disease (CKD). Persistent fibroblast activation and tubular epithelial cell (TEC) injury are key CKD contributors. However, cellular and transcriptional landscapes of CKD and specific activated kidney fibroblast clusters remain elusive. Here, we analyzed single cell transcriptomic profiles of two clinically relevant kidney fibrosis models which induced robust kidney parenchymal remodeling. We dissected the molecular and cellular landscapes of kidney stroma and newly identified three distinctive fibroblast clusters with "secretory", "contractile" and "vascular" transcriptional enrichments. Also, both injuries generated failed repair TECs (frTECs) characterized by decline of mature epithelial markers and elevation of stromal and injury markers. Notably, frTECs shared transcriptional identity with distal nephron segments of the embryonic kidney. Moreover, we identified that both models exhibited robust and previously unrecognized distal spatial pattern of TEC injury, outlined by persistent elevation of renal TEC injury markers including Krt8, while the surviving proximal tubules (PTs) showed restored transcriptional signature. Furthermore, we found that long-term kidney injuries activated a prominent nephrogenic signature, including Sox4 and Hox gene elevation, which prevailed in the distal tubular segments. Our findings might advance understanding of and targeted intervention in fibrotic kidney disease.

Ketamine as a neuroprotective and anti-inflammatory agent in children undergoing surgery on cardiopulmonary bypass: a pilot randomized, double-blind, placebo-controlled trial.

OBJECTIVE: Infants are potentially more susceptible to cell death mediated via glutamate excitotoxicity attributed to cardiopulmonary bypass. We hypothesized that ketamine, via N-methyl D-aspartate receptor blockade and anti-inflammatory effects, would reduce central nervous system injury during cardiopulmonary bypass. METHODS: We randomized 24 infants, without chromosomal abnormalities, to receive ketamine (2 mg/kg, n = 13) or placebo (saline, n = 11) before cardiopulmonary bypass for repair of ventricular septal defects. Plasma markers of inflammation and central nervous system injury were compared at the end of surgery, and 6, 24, and 48 hrs after surgery. Magnetic resonance imaging and spectroscopy before cardiopulmonary bypass and at the time of hospital discharge were performed in a subset of cases and controls (n = 5 in each group). Cerebral hemodynamics were monitored postoperatively using near-infrared spectroscopy, and neurodevelopmental outcomes were assessed using Bayley Scales of Infant Development-II before and 2-3 wks after surgery. RESULTS: Statistically significant differences were noted in preoperative inspired oxygen levels, intraoperative cooling and postoperative temperature, respiratory rate, platelet count, and bicarbonate levels. The peak concentration of C-reactive protein was lower in cases compared to controls at 24 hrs (p = .048) and 48 hrs (p = .001). No significant differences were noted in the expression of various cytokines, chemokines, S100, and neuron-specific enolase between the cases and controls. Magnetic resonance imaging with spectroscopy studies showed that ketamine administration led to a significant decrease in choline and glutamate plus glutamine/creatine in frontal white matter. No statistically significant differences occurred between pre- and postoperative Bayley Scales of Infant Development-II scores. CONCLUSIONS: We did not find any evidence for neuroprotection or neurotoxicity in our pilot study. A large, adequately powered randomized control trial is needed to discern the central nervous system effect of ketamine on the developing brain. brain. TRIAL REGISTRATION: The trial is registered at www.ClinicalTrials.gov, NCT00556361.

Diffusion tensor imaging of white matter injury in a rat model of infantile hydrocephalus.

OBJECTIVE: Diffusion tensor imaging (DTI) is a non-invasive MRI technique that has been used to quantify white matter (WM) abnormality in both clinical and experimental hydrocephalus (HCP). However, no DTI study has been conducted to characterize anisotropic diffusion properties in an animal model of infantile HCP. This DTI study was designed to investigate a rat model of HCP induced at postnatal day 21, a time developmentally equivalent to the human infancy. METHODS: DTI data were acquired at approximately 4 weeks after the induction of HCP with kaolin injection. Using a 7 Tesla small animal MRI scanner we performed high-resolution DTI on 12 rats with HCP and 6 saline controls. Regions of interest (ROI) examined with quantitative comparisons include the genu, body, and splenium of the corpus callosum (gCC, bCC, and sCC, respectively), anterior, middle, and posterior external capsule (aEC, mEC, and pEC, respectively), internal capsule (IC), and fornix (FX). For each ROI, DTI metrics including fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (Dax), and radial diffusivity (Drad) were calculated. RESULTS: We found that the anisotropic diffusion properties were abnormal across multiple WM regions in the brains of the HCP rats. Statistically significant differences included: (1) decreased FA and increased MD and Drad values in the gCC and bCC; (2) increased Dax in the sCC; (3) increased FA and Dax in the aEC; (4) increased FA in the mEC; (5) increased MD and Drad in the pEC; (6) increased FA and Dax in IC; (7) increased FA in FX. CONCLUSIONS: These preliminary results provide the first evidence of WM injury quantified by DTI in a rat model of infantile HCP. Our data showed that DTI is a sensitive tool to characterize patterns of WM abnormalities and support the notion that WM impairment is region specific in response to HCP.

Creatine transporter deficiency impairs stress adaptation and brain energetics homeostasis.

The creatine transporter (CrT) maintains brain creatine (Cr) levels, but the effects of its deficiency on energetics adaptation under stress remain unclear. There are also no effective treatments for CrT deficiency, the second most common cause of X-linked intellectual disabilities. Herein, we examined the consequences of CrT deficiency in brain energetics and stress-adaptation responses plus the effects of intranasal Cr supplementation. We found that CrT-deficient (CrT-/y) mice harbored dendritic spine and synaptic dysgenesis. Nurtured newborn CrT-/y mice maintained baseline brain ATP levels, with a trend toward signaling imbalance between the p-AMPK/autophagy and mTOR pathways. Starvation elevated the signaling imbalance and reduced brain ATP levels in P3 CrT-/y mice. Similarly, CrT-/y neurons and P10 CrT-/y mice showed an imbalance between autophagy and mTOR signaling pathways and greater susceptibility to cerebral hypoxia-ischemia and ischemic insults. Notably, intranasal administration of Cr after cerebral ischemia increased the brain Cr/N-acetylaspartate ratio, partially averted the signaling imbalance, and reduced infarct size more potently than intraperitoneal Cr injection. These findings suggest important functions for CrT and Cr in preserving the homeostasis of brain energetics in stress conditions. Moreover, intranasal Cr supplementation may be an effective treatment for congenital CrT deficiency and acute brain injury.

Diffusion tensor imaging correlates with cytopathology in a rat model of neonatal hydrocephalus.

BACKGROUND: Diffusion tensor imaging (DTI) is a non-invasive MRI technique that has been used to quantify CNS abnormalities in various pathologic conditions. This study was designed to quantify the anisotropic diffusion properties in the brain of neonatal rats with hydrocephalus (HCP) and to investigate association between DTI measurements and cytopathology. METHODS: DTI data were acquired between postnatal day 7 (P7) and P12 in 12 rats with HCP induced at P2 and in 15 age-matched controls. Animals were euthanized at P11 or P22/P23 and brains were processed with immunohistochemistry for glial fibrillary acidic protein (GFAP), ionized calcium-binding adaptor molecule (Iba-1), and luxol fast blue (LFB) to assess astrocytosis, microglial reactivity and degree of myelination, respectively. RESULTS: Hydrocephalic rats were consistently found to have an abnormally low (at corrected p-level of <0.05) fractional anisotropy (FA) value and an abnormally high mean diffusivity (MD) value in the cerebral cortex (CX), the corpus callosum (CC), and the internal capsule (IC). Immunohistochemical analysis demonstrated trends of increasing astrocyte and microglial reactivity in HCP rats at P11 that reached statistical significance at P22/P23. A trend toward reduced myelination in the HCP rats was also found at P22/P23. Correlation analysis at P11 for the CC demonstrated statistically significant correlations (or trends) between the DTI measurement (the decreased FA and increased MD values) and the GFAP or Iba-1 rankings. The immunohistochemical rankings in the IC at P22/P23 were also significantly correlated or demonstrated a trend with both FA and MD values. CONCLUSIONS: This study demonstrates the feasibility of employing DTI on the brain in experimental hydrocephalus in neonatal rats and reveals impairments in multiple regions of interest in both grey and white matter. A strong correlation was found between the immunohistochemical results and the changes in anisotropic diffusion properties.

Role of polyunsaturated fatty acids in human brain structure and function across the lifespan: An update on neuroimaging findings.

There is a substantial body of evidence from animal studies implicating polyunsaturated fatty acids (PUFA) in neuroinflammatory, neurotrophic, and neuroprotective processes in brain. However, direct evidence for a role of PUFA in human brain structure and function has been lacking. Over the last decade there has been a notable increase in neuroimaging studies that have investigated the impact of PUFA intake and/or blood levels (i.e., biostatus) on brain structure, function, and pathology in human subjects. The majority of these studies specifically evaluated associations between omega-3 PUFA intake and/or biostatus and neuroimaging outcomes using a variety of experimental designs and imaging techniques. This review provides an updated overview of these studies in an effort to identify patterns to guide and inform future research. While the weight of evidence provides general support for a beneficial effect of a habitual diet consisting of higher omega-3 PUFA intake on cortical structure and function in healthy human subjects, additional research is needed to replicate and extend these findings as well as identify response mediators and clarify mechanistic pathways. Controlled intervention trials are also needed to determine whether increasing n-3 PUFA biostatus can prevent or attenuate neuropathological brain changes observed in patients with or at risk for psychiatric disorders and dementia.