There are no clinically relevant treatments available that improve function in the growing population of very preterm infants (<32 weeks gestation) with neonatal brain injury. Diffuse white matter injury (DWMI) is a common finding in these children and results in chronic neurodevelopmental impairments1,2. As shown recently, failure in oligodendrocyte progenitor cell maturation contributes to DWMI3. In a previous study, we demonstrated that epidermal growth factor receptor (EGFR) plays an important role in oligodendrocyte development4. Here, we examine whether enhanced epidermal growth factor receptor (EGFR) signaling stimulates the endogenous response of EGFR-expressing progenitor cells during a critical period after brain injury, and promotes cellular and behavioral recovery in the developing brain. Using an established model of very preterm brain injury, we demonstrate that selective overexpression of human (h)EGFR in oligodendrocyte lineage cells or the administration of intranasal heparin binding EGF immediately after injury decreases oligodendroglia death, enhances generation of new oligodendrocytes from progenitor cells (OPCs) and promotes functional recovery. Furthermore, these interventions diminish ultrastructural abnormalities and alleviate behavioral deficits on white matter-specific paradigms. Inhibition of EGFR signaling with a molecularly targeted agent used for cancer therapy demonstrates that EGFR activation is an important contributor to oligodendrocyte regeneration and functional recovery after DWMI. Thus, our study provides direct evidence that targeting EGFR in OPCs at a specific time after injury is clinically feasible and applicable for the treatment of premature children with white matter injury.

Temperature and pH are two of the most important physiological parameters and are believed to be tightly regulated because they are intricately related to energy metabolism in living organisms. Temperature and/or pH data in mammalian brain are scarce, however, mainly due to lack of precise and non-invasive methods. At 11.7T, we demonstrate that a thulium-based macrocyclic complex infused through the blood stream can be used to obtain temperature and pH maps of rat brain in vivo by 1 H chemical shift imaging (CSI) of the sensor itself in conjunction with a multi-parametric model that depends on several proton resonances of the sensor. Accuracies of temperature and pH determination with the thulium sensor -which has a predominantly extracellular presence -depend on stable signals during the course of the CSI experiment as well as redundancy for temperature and pH sensitivities contained within the observed signals. The thulium-based method compared well with other methods for temperature ( 1 H magnetic resonance spectroscopy (MRS) of N-acetyl aspartate and water; copper-constantan thermocouple wire) and pH ( 31 P MRS of inorganic phosphate and phosphocreatine) assessment, as established by in vitro and in vivo studies. In vitro studies in phantoms with two compartments of differing pH values observed under different ambient temperature conditions generated precise temperature and pH distribution maps. In vivo studies in α-chloralose anesthetized and renal-ligated rats revealed temperature (33-34 °C) and pH (7.3-7.4) distributions in the cerebral cortex which are in agreement with observations by other methods. These results demonstrate that the thulium sensor can be used to measure temperature and pH distributions in rat brain in vivo simultaneously and accurately with 1 H CSI.

-/-bone marrow chimeras (BMCs) at days 7 and 15 after ICH. Right: Quantification of residual hematoma volume in the WT and Ccr2 -/-BMCs. n = 11 at day 7; n = 9 at day 15. *P < 0.05 by Student's t test. (B) Cylinder test and apomorphine turning test from WT and Ccr2 -/-BMCs at day 15 after ICH. n = 6/group for cylinder test; n = 8/ group for apomorphine turning test. *P < 0.05 by Student's t test. (C) Cylinder test, neurological deficit score, and corner test in control-and anti-CCR2 antibody-treated mice at days 1 and 3 after collagenase ICH. n = 7/group. *P < 0.05 by 1-way repeated-measures ANOVA and Bonferroni's post hoc test. (D) Top: Representative coronal sections show hematoma from control-and anti-CCR2 antibody-treated WT mice after blood injection ICH at 7 days. Bottom: Quantification of hematoma volume, n = 3/ group. *P < 0.05 versus control by Student's t test. (E) Top: Representative coronal sections show hematoma in the isotype control-and anti-CCR2 antibody-treated mice from collagenase model at day 12. Bottom: Quantification of hematoma volume. n = 8/group. *P < 0.05 versus control by Student's t test. (F) The cylinder test, neurological deficit score, and corner test in isotype control-and anti-CCR2 antibody-treated ICH mice at days 3, 5, 7, 9, and 11 after collagenase ICH surgery. n = 8/group. *P < 0.05 versus isotype control group by 1-way repeated-measures ANOVA and Bonferroni's post hoc test. αCCR2, anti-CCR2 antibody.

The human cerebral cortex is distinguished by its large size and abundant gyrification, or folding, yet the evolutionary mechanisms driving cortical size and structure are unknown. While genes essential for cortical developmental expansion have been identified from the genetics of human primary microcephaly (“small head”, associated with reduced brain size and intellectual disability)1, studies of these genes in mice, whose smooth cortex is one thousand times smaller than that of humans, have provided limited insight. Mutations of abnormal spindle-like microcephaly-associated (ASPM), the most common recessive microcephaly gene, reduce cortical volume by ≥50% in humans2–4, but have little effect in mice5–9, likely reflecting evolutionarily divergent functions of ASPM10,11. We used genome editing to create a germline knockout (KO) of Aspm in the ferret (Mustela putorius furo), a species with a larger, gyrified cortex and greater neural progenitor cell (NPC) diversity12–14 than mice, and closer Aspm protein sequence homology to human. Aspm KO ferrets exhibit severe microcephaly (25–40% decreases in brain weight), reflecting reduced cortical surface area without significant change in cortical thickness, as in human patients3,4, suggesting loss of “cortical units”. The mutant ferret fetal cortex displays a massive premature displacement of ventricular radial glial cells (VRG) to the outer subventricular zone (OSVZ), where many resemble outer radial glia (ORG), an NPC subtype essentially absent in mice and implicated in cerebral cortical expansion in primates12–16. These data suggest an evolutionary mechanism whereby Aspm regulates cortical expansion by controlling the affinity of VRG for the ventricular surface, thus modulating the ratio of VRG, the most undifferentiated cell type, to ORG, a more differentiated progenitor.

Chemical shifts of complexes between paramagnetic lanthanide ions and macrocyclic chelates are sensitive to physiological variations (of temperature and/or pH). Here we demonstrate utility of a complex between thulium ion (Tm 3+ ) and the macrocyclic chelate 1,4,7,10-tetramethyl 1,4,7,10-tetraazacyclodoecane-1,4,7,10-tetraacetate (or DOTMA 4− ) for absolute temperature mapping in rat brain. Feasibility of TmDOTMA − is compared with that of another Tm 3+ -containing biosensor which is based on the macrocyclic chelate 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonate) (or DOTP 8− ). In general, the in vitro and in vivo results suggest that Biosensor Imaging of Redundant Deviation in Shifts (BIRDS) which originate from these agents (but exclude water) can provide temperature maps with good accuracy. While TmDOTP 5− emanates three major distinct proton resonances which are differentially sensitive to temperature and pH, TmDOTMA − has a dominant pH-insensitive proton resonance from a −CH 3 group to allow higher signal-to-noise ratio (SNR) temperature assessment. Temperature (and pH) sensitivities of these resonances are practically identical at low (4.0T) and high (11.7T) magnetic fields and at nominal repetition times only marginal SNR loss is expected at the lower field. Since these resonances have extremely short relaxation times, high-speed chemical shift imaging (CSI) is needed to detect them. Repeated in vivo CSI scans with BIRDS demonstrate excellent measurement stability. Overall, results with TmDOTP 5− and TmDOTMA − suggest that BIRDS can be reliably applied, either at low or high magnetic fields, for functional studies in rodents.

Energetics of resting and evoked fMRI signals were related to localized ensemble firing rates () measured by electrophysiology in rats. Two different unstimulated, or baseline, states were established by anesthesia. Halothane and ␣-chloralose established baseline states of high and low energy, respectively, in which forepaw stimulation excited the contralateral primary somatosensory cortex (S1). With ␣-chloralose, forepaw stimulation induced strong and reproducible fMRI activations in the contralateral S1, where the ensemble firing was dominated by slow signaling neurons (SSN; range of 1-13 Hz). Under halothane, weaker and less reproducible fMRI activations were observed in the contralateral S1 and elsewhere in the cortex, but ensemble activity in S1 was dominated by rapid signaling neurons (RSN; range of 13-40 Hz). For both baseline states, the RSN activity (i.e., higher frequencies, including the ␥ band) did not vary upon stimulation, whereas the SSN activity (i.e., ␣ band and lower frequencies) did change. In the high energy baseline state, a large majority of total oxidative energy [cerebral metabolic rate of oxygen consumption (CMR O2)] was devoted to RSN activity, whereas in the low energy baseline state, it was roughly divided between SSN and RSN activities. We hypothesize that in the high energy baseline state, the evoked changes in fMRI activation in areas beyond S1 are supported by rich intracortical interactions represented by RSN. We discuss implications for interpreting fMRI data where stimulus-specific ⌬CMR O2 is generally small compared with baseline CMR O2.awake ͉ behavior ͉ calibrated fMRI ͉ glucose ͉ glutamate N oninvasive NMR and electrophysiological methods offer considerably different spatiotemporal results that presumably reflect the same cerebral activity. Localized energy consumption of neuronal and glial populations in MRI voxels has been evaluated (1), initially from 13 C MRS (2) and more recently from calibration of functional MRI (fMRI) (3). In vivo electrophysiological measurements of neuronal activity, from single neurons or large ensembles (4), are considered the gold standard of cerebral activity (5). Can measurements from these dissimilar techniques provide complementary insights into the working brain?A promising convergence between these apparently different results relies on a universal thermodynamic principle, the fundamental relationship between the work done and the energy expended. Cerebral energy comes almost exclusively from glucose oxidation (6). Recent results have shown that the cerebral metabolic rate of oxygen consumption (CMR O2 ) is almost completely dedicated to supporting work associated with synaptic activity (7,8). Changes in CMR O2 from calibrated fMRI (9) are linear with changes in firing rates of a representative neuronal ensemble in the same voxel (10). This basic work/ energy relationship has been extended by in vivo investigations (11, 12) that relate imaging energetics to the underlying neuronal activities.Neuroimaging methods localize changes of task-i...

The opening of basepairs plays a key role in DNA replication and transcription, and in the action of DNA repair and modification enzymes. In this article, we have used proton exchange to define the energetics of the pathways for basepair opening in two DNA 17-mer duplexes. The rates of exchange of imino protons with solvent protons were measured by NMR spectroscopy for each DNA duplex, as a function of the concentration of exchange catalyst and of temperature. The measurements provided the rates and the equilibrium constants of the opening reactions for individual basepairs at different temperatures. These temperature dependences were used to calculate the enthalpies and the free energies of the barrier to opening and of the open state for each basepair. The results reveal the existence of three distinct patterns of enthalpy changes in the opening reactions. The patterns differ from each other in the location of the kinetic opening barrier relative to the open state. Neighboring bases, which are one or more positions removed from the opening basepair, influence the enthalpic pattern of the opening pathway. The free energies of the opening barriers are found to be linearly related to the free energies of the open state. This correlation is analyzed in terms of rate-equilibrium free energy relationships previously observed in other systems, and suggests that the transition state in the opening reaction is closer to the native closed state of the basepair than to its open state.

Solid tumors have acidic extracellular pH (pHe) but near neutral intracellular pH (pHi). Because acidic pHe milieu is conducive to tumor growth and builds resistance to therapy, simultaneous mapping of pHe inside and outside the tumor (i.e., intratumoral-peritumoral pHe gradient) fulfills an important need in cancer imaging. We used Biosensor Imaging of Redundant Deviation in Shifts (BIRDS), which utilizes shifts of nonexchangeable protons from macrocyclic chelates (e.g., 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonate) or DOTP8−) complexed with paramagnetic thulium (Tm3+) ion, to generate in vivo pHe maps in rat brains bearing 9L and RG2 tumors. Upon TmDOTP5− infusion, MRI identified the tumor boundary by enhanced water transverse relaxation and BIRDS allowed imaging of intratumoral-peritumoral pHe gradients. The pHe measured by BIRDS was compared with pHi measured with 31P-MRS. In normal tissue pHe was similar to pHi, but inside the tumor pHe was lower than pHi. While the intratumoral pHe was acidic for both tumor types, peritumoral pHe varied with tumor type. The intratumoral-peritumoral pHe gradient was much larger for 9L than RG2 tumors, because in RG2 tumors acidic pHe was found in distal peritumoral regions. Increased presence of Ki-67 positive cells beyond the RG2 tumor border suggested that RG2 was more invasive than 9L tumor. These results indicate that extensive acidic pHe beyond the tumor boundary correlates with tumor cell invasion. In summary, BIRDS has sensitivity to map in vivo intratumoral-peritumoral pHe gradient, thereby creating preclinical applications in monitoring cancer therapeutic responses (e.g., with pHe-altering drugs).