Membrane depolarization and aberrant lipid distributions in the neonatal rat brain following hypoxic-ischaemic insult OPEN
2018

Dominika Luptakova
Institute of Microbiology
Czech Academy of Sciences
142 20PragueCzech Republic

Institute of Experimental Pharmacology and Toxicology
CEM of the SAS
841 04BratislavaSlovakia

Ladislav Baciak
Institute of Experimental Pharmacology and Toxicology
CEM of the SAS
841 04BratislavaSlovakia

Central Laboratories
Slovak University of Technology
812 37BratislavaSlovakia

Tomas Pluhacek
Institute of Microbiology
Czech Academy of Sciences
142 20PragueCzech Republic

Department of Analytical Chemistry
Regional Centre of Advanced Technologies and Materials
Olomouc

Anton Skriba
Institute of Microbiology
Czech Academy of Sciences
142 20PragueCzech Republic

Blanka Sediva
Institute of Microbiology
Czech Academy of Sciences
142 20PragueCzech Republic

Vladimir Havlicek
Institute of Microbiology
Czech Academy of Sciences
142 20PragueCzech Republic

Department of Analytical Chemistry
Regional Centre of Advanced Technologies and Materials
Olomouc

Ivo Juranek
Institute of Experimental Pharmacology and Toxicology
CEM of the SAS
841 04BratislavaSlovakia

Membrane depolarization and aberrant lipid distributions in the neonatal rat brain following hypoxic-ischaemic insult OPEN

ScIentIfIc RePoRtS |
86952201810.1038/s41598-018-25088-2Received: 29 December 2017 Accepted: 13 April 20181 771 47, Czech Republic. Correspondence and requests for materials should be addressed to V.H. (email: firstname.lastname@example.com)
Neonatal hypoxic-ischaemic (HI) encephalopathy is among the most serious complications inneonatology. In the present study, we studied the immediate (0 hour), subacute (36 hours) and late (144 hours) responses of the neonatal brain to experimental HI insult in laboratory rats. At the striatal level, the mass spectrometry imaging revealed an aberrant plasma membrane distribution of Na + /K + ions in the oedema-affected areas. The failure of the Na + /K + gradients was also apparent in the magnetic resonance imaging measurements, demonstrating intracellular water accumulation during the acute phase of the HI insult. During the subacute phase, compared with the control brains, an incipient accumulation of an array of N-acylphosphatidylethanolamine (NAPE) molecules was detected in the HI-affected brains, and both the cytotoxic and vasogenic types of oedema were detected. In the severely affected brain areas, abnormal distributions of the monosialogangliosides GM2 and GM3 were observed in two-thirds of the animals exposed to the insult. During the late stage, a partial restoration of the brain tissue was observed in most rats in both the in vivo and ex vivo studies. These specific molecular changes may be further utilized in neonatology practice in proposing and testing novel therapeutic strategies for the treatment of neonatal HI encephalopathy.Published: xx xx xxxx OPEN www.nature.com/scientificreports/

Neonatal hypoxic-ischaemic encephalopathy (HIE) is among the most common neurological complications in neonatology. Due to its high mortality and chronic morbidity in surviving infants, HIE represents a serious socioeconomic issue 1 . The incidence of HIE is 1-4 per 1000 live births in developed countries and is usually higher in developing countries 2 . In approximately 25% of survivors, hypoxic-ischaemic (HI) brain injury can result in a variety of severe neuro-psychological symptoms, such as cerebral palsy, seizures, epilepsy, mental retardation, learning and/or behavioural disorders, etc 3 . The early phase of HI brain insult is characterized by primary energy metabolism failure caused by an impaired cerebral blood flow and insufficient oxygen supply 4 . After the re-oxygenation of the brain and the ostensible recovery of its functions, secondary energy failure may occur. The secondary injury is considered responsible for the long-lasting consequences of HI insult 5 . At the beginning of HI insult, the brain metabolism switches to the anaerobic mode due to the low oxygen availability. The inhibition of oxidative phosphorylation leads to a decrease in the phosphocreatine and ATP levels and an increase in the inorganic phosphate and lactic acid levels. The hypoxic inhibition of ATP-dependent ion pumps leads to cell membrane depolarization, which is mostly due to potassium ion leakage from the cell and electrochemically favoured Na + and Ca 2+ influx into the cell. This depolarization, in turn, leads to water entry and accumulation inside the cell and, thus, cell swelling. The cell swelling along with the cytosolic calcium overload may ultimately result in cell lysis 6,7 . Hence, the water accumulation inside the cell underlies the evolution of cytotoxic oedema, which is an early sign of HI insult 8 .

During the reperfusion/re-oxygenation phase, the brain metabolism may appear to recover. The hypoxia-induced cytosolic calcium overload may diminish due to increased calcium sequestration inside ScIentIfIc RePoRtS | (2018) 8:6952 | DOI: 10.1038/s41598-018-25088-2 intracellular organelles, such as the endoplasmic reticulum and mitochondria. However, this sequestration may lead to calcium overload in these organelles, resulting in their dysfunction. Moreover, the reintroduction of oxygen into posthypoxic tissue can result in the overproduction of reactive oxygen and nitrogen species that may exacerbate the primary HI injury 9 . Due to the mitochondrial calcium overload, the mitochondria may functionally and structurally disintegrate, resulting in secondary energy metabolism failure 4,7,10 , and the activation of calcium-dependent phospholipases, proteases and endonucleases and the resulting lipid peroxidation, protein oxidation, and fragmentation of DNA/RNA are responsible for triggering cell death processes 6,10,11 . The overall process is characterized by excitotoxicity, progressively evolving brain oedema and exacerbation of the primary insult. The HI-induced break-up of cellular membranes causes the release of membrane-derived lipid metabolites 12 .

For a timely diagnosis of neonatal brain HI insult and the application of effective therapeutic interventions, the period between the primary and secondary energy failures is crucial. This period is generally referred to as "the window of opportunity" 13 . In neonatology practice, newborns who meet the inclusion criteria (i.e., gestation age ≥ 36 weeks, Apgar score < 5 at 10 minutes, severe acidosis (pH < 7), umbilical cord base deficit ≥16 mmol/L and signs of foetal distress/acute perinatal events) should be considered for mild hypothermia (32-34.5 °C) therapy, which is the only currently approved therapy. However, therapeutic hypothermia is not very effective [14][15][16] . Only one in every 8-9 HI-affected newborns treated with therapeutic hypothermia survives without moderate or severe consequences, and up to half of all hypothermia-treated infants may suffer permanent neurological consequences 17 .

In the present study, we focused on cellular and molecular processes occurring in acute, subacute and late stages of HI brain insult in newborn rats. While searching for potential predictive biomarker(s) of the neonatal brain HI injury, we particularly focused on brain lipidome by means of multimodal in vivo magnetic resonance imaging (MRI), ex vivo light microscopy and mass spectrometry imaging (MSI) techniques. MSI combined matrix-assisted laser desorption/ionization (MALDI) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). We report a close association between the early evolution of oedema and an aberrant Na + and K + distribution, reflecting cell membrane depolarization. Importantly, we identified distinct lipid metabolites that could be used prospectively as biomarkers reflecting the severity of neonatal HIE.

Results and Discussion

In vivo monitoring of the acute, subacute and late stages of HIE. After HI insult, the HI-affected rats curved to the left side from the central body axis as an immediate response to the insult, while the non-affected sham control rats remained in an apparently normal position (Fig. 1A). The development of cerebral oedema was monitored in vivo by diffusion-weighted (DW) magnetic resonance imaging (DWI). Cytotoxic oedema affected the cortex, corpus callosum, external capsule, striatum, hippocampus, hypothalamus, thalamus, lateral septal nuclei and amygdala (Supplementary Figure S1D). The average size of the cytotoxic oedema-affected area in the ipsilateral hemisphere (seven biological replicates) was 11.9 ± 1.2% (mean ± standard error of the mean (SEM)) ( Supplementary Table S1). Intriguingly, in two HI-insulted rats, the cytotoxic oedema spread to the contralateral hemisphere, mainly affecting the cortex, and represented 1.7 ± 1.2% of the total hemisphere size (Supplementary Figure S1B). Cytotoxic oedema lesions with the typical massive intracellular water accumulation provided bright hyperintensive signals on DWI (Fig. 1B). The 3D reconstruction was built from consecutive slices (Fig. 1C). No oedema development was observed in the control animals (n = 6).

At 36 hours after HI insult, the cytotoxic oedema size significantly decreased, and cytotoxic oedema-related hyperintensities were observed only in the cortex of the ipsilateral hemisphere. However, the hypointensities characteristic of the vasogenic type of oedema began to appear in the lateral ventricle, hippocampus and external capsule. At this time point, both the cytotoxic and vasogenic types of oedemas were simultaneously present, which is referred to as biphasic oedema (Fig. 1D). In the sham animals, no pathological changes were observed on DWI, and both hemispheres had an identical appearance.

During the late stage of the HI insult (144 hours), only two of the seven surviving rats developed progression of the brain lesion as documented by the larger extent of the vasogenic oedema, which was indicative of a negative outcome for these animals. An increased hypointensive signal was observed in the lateral ventricle, hippocampus, striatum, corpus callosum, external capsule and cortex brain regions. No characteristic changes were observed in the other five rats, and their initial brain lesions decreased to the zero level (Supplementary Figure S1C).

Cell membrane depolarization as an immediate response to HI insult monitored ex vivo.

The ex vivo changes in neuronal vacuolization were detected by microscopy as numerous shapeless and optically empty vacuoles in the cytoplasm and were observed primarily in the cortex during the early stage of HI insult ( Fig. 2A). Cytotoxic oedema in the neuronal cells manifested as a triangular nuclei shape, nuclear membrane thickening and neuronal swelling (Fig. 2Aa). No traces of neuronal cell death were observed. In all HI brain samples, signs of oedema were detected in the cortex, striatum, external capsule and corpus callosum of the ipsilateral hemisphere. In four HI samples, oedema spread to the contralateral hemisphere, primarily affecting the cingulate cortex, motor cortex and corpus callosum. Compared to the control animals, a visible compression of the ventricular system was typically observed in all HI brains. Notably, the non-affected areas possessed features that were similar to those in the control sham brains.

Phosphatidylcholines (PCs) dominated the positive ion mode MALDI spectra of both the HI-insulted (Supplementary Figure S2A)  HI-affected brains. The distribution of PC (16:0/16:0) is depicted in Fig. 2B and D. The structure of the lipids was acquired by collision-induced dissociation MS analysis (see Supplementary Figure S3 as an example).

The molecular profiles of the lipids cationized with Na + or K + matched well with the elemental profiles of sodium and potassium. According to the quantitative LA-ICP-MSI analysis, the sodium concentration was approximately 1.5-fold higher at the striatal level of the HI-affected brain area (Fig. 2C) and the potassium concentration was significantly lower (Fig. 2E) than the uniform, symmetric distribution of these elements in the control brain. In the LA-ICP-MSI analysis, a sufficient linear range was achieved for both the sodium and potassium calibration plots using dried droplet calibration standards (Supplementary Figure S4).

The most abundant signals in the negative ion MALDI mass spectra were attributed to phosphoethanolamines (PE), phosphatidic acids (PA), phosphoinositoles (PI) and monosialoganglioside GM1(d18:1/18:0) (m/z 1544.863) (Supplementary Figure S2B). The most intense peak observed in all spectra occurred at m/z 885.549 and was ascribed to PI (38:4). The phosphate-containing molecules ADP (m/z 426.021) and AMP (m/z 346.053) were detected as [M-H] − ions, and their structures were assigned using both exact mass and tandem mass spectrometry (Supplementary Figures S5 and S6). In the m/z mass range from 970 to 1070, an array of N-acylphosphatidylethanolamine (NAPE) molecules was detected in the spectra of the HI-affected brains. During the acute stage following HI insult, the relative abundance of the NAPEs was approximately 2-fold higher in the affected animals than that in the control sham brains (n = 6).

Subacute changes in the brain lipidome. The subacute response was analysed at the 36-hour time interval and involved significant damage to neuronal tissue, with signs of liquefactive necrosis resulting in the occurrence of large-body astrocytes with enlarged vesicular nuclei, an abundant eosinophilic cytoplasm and neuronal debris (Fig. 3A). The myelin in the necrotic area appeared pale due to the surrounding oedema fluid, and many macrophages were present. The necrotic area was nearly identical to the actual ischaemic core of the lesion, which was surrounded by penumbra comprising shapeless and blobbing neurons and/or neurons undergoing necrotic cell death. The severe lesions were predominantly located in the ipsilateral hemisphere of the HI-insulted brain, and the ischaemic core typically affected the motor cortex, somatosensory cortex, external capsule and upper portion of the striatum. In the corpus callosum, the characteristic initial stage of cell degeneration was observed as pale or slightly pink tissue. In the contralateral hemisphere, the corpus callosum and external capsule were the only structures affected.

The results of the positive ion mode MALDI MSI measurements were similar to those observed during the acute stage of HI injury. Aberrant distribution of Na + and K + adducts of the PCs was detected in the severely HI-affected brain areas, where the Na + adducts of the PCs dominated the analogous K + adducts. Thus, the persistent membrane depolarization was associated with the accumulated oedema fluid (Supplementary Figure S7).

The negative ion MALDI MSI revealed that the HI-insulted brain significantly differed from the sham brain. Aberrant distribution of NAPEs, GM2, and GM3 (Supplementary Table S3) was found in four of the six HI-insulted brains (Fig. 4B). Importantly, these aberrations corresponded to the severely degenerated tissue of the ischaemic core affecting the motor, somatosensory cortex and upper portion of the striatum (Fig. 3B,C). Moreover, areas with increased levels of NAPEs, GM2, and GM3 matched those that had a decreased concentration of high-energy phosphate compounds (Supplementary Figure S7).

An apparent brain recovery was observed in two other HI-insulted rats. This finding was attributed to the biological variability in the rat pups exposed to moderate HI insult, particularly their exposure to hypoxia under mild hypothermia (34 °C). Modifying the original experimental model was important because the 36.5 °C conditions used previously resulted in a profound tissue reduction in the affected hemisphere and an overall mortality rate of 22% (data not shown). In the present study, this modification enabled us to follow the molecular changes in the HI-affected tissue, which still possessed sufficient integrity for the MSI application. Importantly, NAPEs, GM2, and GM3 were also present as endogenous molecules in the control brain; however, their distribution was uniform, and their concentration was markedly low (Supplementary Figure S7). Similar to NAPEs, GM2 (d18:1/18:0) and GM3 (d18:1/18:0) were found in their deprotonated forms at m/z 1382.805 and m/z 1179.736, respectively, and were markedly increased in the HI-affected areas (Fig. 4). The GM2 ganglioside molecule was characterized according to the appropriate MS/MS fragmentation spectrum, showing characteristic neutral losses (Supplementary Figure S8). GM2 and GM3 showed a spatial distribution similar to that of NAPE species. Moreover, their population increased specifically in the area of the penumbra and spread to the insular cortex, piriform cortex, corpus callosum and external capsule.

The profiles of the most abundant NAPE representative (54:4), GM2 and GM3 are shown in Fig. 5. A somatosensory cortex area representing approximately 260-300 pixels was selected for the semi-quantitative statistical assessment, and the relative intensities of the [M-H] − ions were normalized to PI (38:4), which represents the base peak at m/z 885 in all MALDI mass spectra. The statistical analysis of the differences between the HI and sham groups was performed using the non-parametric two-tailed Mann-Whitney U test. Statistical significance at a p-value < 0.05 (*p) was obtained for the sum of all NAPEs, GM2, and GM3. The same significance (*p < 0.05) was also obtained for NAPE (52:4), NAPE (54:4) and NAPE (58:6). Regarding GM3, the statistical significance was even higher, with a p-value equal to 0.004 (Supplementary Table S4). Regarding GM3, the statistical significance was also confirmed by  Table S5). For the complete results, see the statistical analysis in the Materials and methods section.

Notably, in the semi-acute stage of the neonatal rat brain HI injury, we found remarkably increased NAPE, GM2 and GM3 levels in HI affected regions. In general, these compounds are important lipid mediators involved in early brain development and during injurious processes in the adult brain 19 . Our findings on the accumulation of NAPEs in the HI affected neonatal rat brain areas starting from 0 hour and peaking at 36 hours of the HI insult are in good agreement with the reports on markedly increased levels of NAPEs in the brain of adult rats and mice 5 and 24 hours after focal cerebral ischemia induction, respectively 20 . These data indicate involvement of NAPEs in the acute and semi-acute phase of HI brain injury. Importantly, NAPEs accumulation due to post-decapitative ischaemia was high in the brain of newborn rats, yet relatively low in adolescent and adult rats, indicating the significance of NAPEs in the developing brain 19 . NAPEs are precursors of N-acylethanolamines (NAEs), bioactive lipids with anti-inflammatory and neuroprotective effects, in particular protecting neurons from glutamatergic excitotoxicity 21 . Some of NAEs are endocannabinoids, e.g. anandamide (N-arachidonoylethanolamine), operating via the cannabinoid CB1/CB2 receptors and providing neuroprotection, including that from ischaemic damage 22,23 . In the present study, the increase of NAPEs in the affected brain areas observed at 36 hours of HI insult may reflect a tendency of the brain to limit the evolving HI injury.

Regarding the role of gangliosides in the developing brain, they are known to be synthesized and immediately metabolized in postnatal age. Our findings of low GM2/GM3 levels in the control brain of 7-day old rats are in agreement with the previous statement. Gangliosides were reported to accumulate during progressive neuronal damage 24 . Herein, we detected no increase in either GM2 or GM3 in the HI affected brain at 0 hour of the HI insult. On the other hand, their levels markedly increased at 36-hour time interval indicating the significance of GM2/GM3 in the semi-acute stage of neonatal brain HI injury. Whether the increased GM2/GM3 level reflects an effort of the HI brain to self-protect similarly to that of NAPE, this observation needs to be further elucidated.

Compensation mechanism during the late stage after HI insult. We observed an ongoing restoration of the HI-affected brain tissue, and a gradual recovery of the overall brain architecture was observed on the sixth day (144 hours) after the HI insult. The cellular morphology, neurons and astrocytes in the HI-affected brain areas were not completely recovered as demonstrated by the distorted shape of the neuronal cells, the foamy appearance of the cytoplasm and signs of blobbing. The formation of a "halo" sign was observed around the neuronal cells. Angiogenesis, reflecting tissue compensation for the lack of oxygen, was observed. Despite these  Figure S9).

In contrast, considerable tissue loss was detected by MRI during the late stage of the insult. The overall tissue reduction in the HI-affected brains, as assessed by the ipsilateral/contralateral hemisphere volume ratio, was 13.1 ± 1.4% (mean ± standard error of the mean (SEM)) in a set of seven animals (Supplementary Table S6). In an analogous set of control animals (n = 6), the tissue loss was marginal. Similarly, no changes were observed between the HI-insulted and control sham rat brains at the molecular level using positive ion mode MALDI MSI measurements, while a slight persistently increased intensity of the monosialoganglioside GM3 was determined using the negative ion mode (four animals). No significant changes were observed in the levels of NAPEs or GM2 species. The multiple parametric and non-parametric testing of the time effect on the levels of NAPEs, GM2 and GM3 revealed statistically significant differences between times 0 and 36 hours for GM2 and GM3, and the respective p-values were 0.003 and 0.013 in the parametric tests and 0.046 and 0.039 in the non-parametric tests (see Supplementary Tables S7 and S8, respectively). Wistar rat females and males (both from the breeding station in Dobra Voda by Trnava, Slovak Republic) were mated at a 3:1 ratio for 6 days, separated and allowed to give birth at term. At the age of 7 days, the development and maturation of the rat brain are histologically similar to those in near-term human infants (32-36-week of gestation), and the typical characteristics include complete cortical neuronal layering, regression of germinal matrix and partial myelination 25 . Seven-day-old male pups (weighing 13-18 g) were anesthetized with isoflurane (4% for induction and 2% for maintenance), and the left common carotid artery was ligated with surgical silk 26 . Male pups were selected for the experiment because their sensitivity to hypoxic-ischaemic damage is known to be higher than that of female pups 27 . The neck incision was sutured, and the pups were allowed to recover from the anaesthesia for one hour. Subsequently, the pups were exposed to hypoxia (8% oxygen conditions, achieved by mixing air with gaseous nitrogen) in a sealed chamber at 34 °C for 90 minutes. The sham (control) animals were subjected to anaesthesia, surgical neck incision and common left carotid artery isolation but did not undergo ligation. After 1-hour recuperation, the sham pups were placed in a chamber with normoxic atmosphere at 34 °C for 90 minutes.

The overall experimental scheme consisted of two parallel experiments, i.e., in vivo and ex vivo. The brain changes at three time intervals after HI insult were assessed. In the in vivo experiments, the HI-affected (n = 9) and sham (n = 6) rat pups were allowed to survive, and MRI data were collected at 0-hour, 36-hour and 144-hour time intervals. Two HI-insulted rats did not survive to the sixth (144 h) day and were excluded from the experiment. In the ex vivo experiments, three sets of HI-affected and sham pups were sacrificed by decapitation at the 0-hour (n = 6/6, HI-affected/sham), 36-hour (n = 6/6) and 144-hour (n = 4/4) time intervals to assess the acute, subacute or late phase brain damage. All brains were quickly removed from the skull, snap-frozen in liquid nitrogen and stored at −80 °C until further analyses.

Cryosectioning and histology. Prior to sectioning, the deeply frozen brain samples were allowed to warm at −11 °C in a Leica cryostat CM1950 (Wetzlar, Germany) chamber. The striatal level was located using a stereotaxic atlas 28   width were used, respectively. The sections were thaw-mounted onto precooled ITO glass slides for the MALDI MSI and light microscopy analyses. For the LA-ICP-MS analyses, the sections were directly deposited onto an Omni Slide without thawing. All slides were vacuum-dried in a desiccator at room temperature for 40 minutes prior to further use.

For histology evaluation, the slices were rehydrated in descending aqueous ethanol concentrations (100%, 95% and 80%) and stained with haematoxylin, Gill No. 2 (Sigma Aldrich, Prague, Czech Republic) to visualize the cellular nuclei. Then, the slide was flushed with water, and the excessive colorant was removed by acidic ethanol. Eosin counterstaining was subsequently performed. Tissue slices were serially dehydrated in ethanol solutions (95% and 100%), toned with xylene and permanently fixed with DPX mounting medium. Finally, the slides were examined as light-microscopic images using an Axio Scan.Z1 slide scanner (Zeiss, Jena, Germany) and processed using ZEN 2.3 Lite (Zeiss, Jena, Germany) software.

MALDI mass spectrometry profiling and imaging. MALDI MS was performed using a 12 T SolariX FTICR mass spectrometer (Bruker Daltonics, Billerica, USA). CHCA (7 mg/mL in 50% ACN/0.1% TFA) and 9-AA (7 mg/mL in 75% MeOH) matrices were applied to the tissue attached onto the ITO glass slide using ImagePrep (Bruker Daltonics, Bremen, Germany, default method for CHCA). The product ion mass spectra were collected at 3 Da isolation width and a 30 V collision energy and processed using DataAnalysis 4.2 (Bruker Daltonics, Bremen, Germany). Desorption from the tissue was performed using laser attenuations of 22 or 26%. In the MS imaging experiments, the lipids and metabolites were ionized/desorbed using a 100-µm raster step size with a SmartBeam II laser (2 kHz, 200 shots/pixel) and 16,000 resolution (FWHM) at m/z 400. The raw MSI data were analysed using FlexImaging 4.1 (Bruker Daltonics, Bremen, Germany), transformed into an imzML file and processed using CycloBranch 29 .

Quantitative laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). An

Analyte G2 LA unit (Photon Machines, Redmont, USA) equipped with an ArF excimer nanosecond laser (193 nm) was used to perform the tissue ablation. The coupling of the LA and 7700× ICP-MS (Agilent Technologies, Tokyo, Japan) units was achieved with Tygon ® (1.2 m × 4 mm) tubing. The raw data were imported as CSV files to ImageLab multisensor imaging software 30 , and the constructed distribution maps were correlated with the corresponding histological images. Details regarding the calibration plots and quantification are provided in Supplementary Figure S4.

The quantification experiments were performed using a set of dried droplet calibration standards as previously described 31,32 . Briefly, filter paper discs with a 5-mm diameter (Whatman 589/1, ashless) were placed onto a microscope glass slide using double-sided tape and spiked with 10 μL of a liquid standard solution with increasing concentrations of sodium and potassium (0, 10, 100, 500, 1000, and 4000 μg.mL −1 Na and K in 1% HNO 3 ). Blank "calibration" was performed under the same conditions using 10 μL of 1% HNO 3 . After applying the liquids to the filter targets, the standards were allowed to dry at room temperature overnight in a Holten safe 2010 1.2 laminar flow cabinet (Live Technologies, Prague, Czech Republic).

Magnetic resonance imaging. Each examined animal was placed onto a dedicated holder, and inhalation anaesthesia (2% isoflurane in air) was applied while the respiration rate was maintained within the 50-70 BPM range. All MRI experiments were performed at 37 °C using a 4.7 T direct-drive horizontal scanner (Agilent, Yarton, UK) equipped with a 400-mT/m gradient and 120-mm bore diameter insert. A Rapid Biomed (Rimpar, Germany) quadrature volume coil transmitter with an i.d. of 72 mm and a STARK Contrast (Erlangen, Germany) surface coil receiver were used for the signal detection. A diffusion-weighted sequence (FSEMSDW) was used with the following parameters: TR/TE eff = 3000/36 ms, b-value = 1000 s/mm 2 , echo train length = 8, echo spacing = 10 ms, 10 averages, FOV = 25 × 25 mm 2 , and matrix size = 128 × 128. Eighteen contiguous 1-mm thick coronal slices were scanned to cover the brain regions of interest. The overall scan time was 8 min.

Both the data processing and volumetry were performed using ImageJ software, version 1.47 33 . A median filter with a 2-pixel radius was applied to reduce the noise and delineate the boundaries of the image. The overall lesion size was calculated by summing the hyperintensive area of all slices and multiplying by the slice thickness. Both the ipsilateral and contralateral lesion volumes were divided by the appropriate hemisphere volume. The measurement reliability was determined at the level of the slice for the oedema (n = 88) and brain (N = 108) measurements by evaluating the intra-class correlation coefficient using MedCalc for Windows, version 16.4 (MedCalc Software, Ostend, Belgium).

Statistical analysis. The variability in the relative abundance of NAPEs, GM2, and GM3 in the technical replicates was measured by the coefficient of variation (CV), which is defined as the ratio of the standard deviation (SD) to the arithmetic mean (Supplementary Tables S9 and S10). The data are presented as the mean and SEM, and the differences between groups are presented as standard box-and-whisker diagrams with outliers plotted as individual points (Supplementary Figs S10-S16). The technical variability was less than 0.097 for NAPEs and GM2 and less than 0.135 for GM3. The analysis of variability demonstrated that the main component influencing the variability is biological variability. The percentage of biological variability in the total variability was within the ranges of 90.2-99.8%, 80.9-95.3%, and 39.1-93.2% for NAPEs, GM2, and GM3, respectively (Supplementary Table S11).

The biological variability was demonstrated by the coefficient of variation and variance decomposition using one-way ANOVA (Supplementary Table S7). The Gaussian distribution of the data sets was assessed by performing Lilliefors corrected Kolmogorov-Smirnov tests (Supplementary Table S12). The mean and SEM of the relative abundance of the NAPE representatives, GM2 and GM3 at the 0-, 36 non-parametric testing approaches were applied. The statistical significance of the differences between the mean values of the HI-insulted group and control group was tested by performing a two-tailed t-test (Supplementary  Table S5). The equivalence test of medians was based on 95% confidence intervals calculated using a Mann-Whitney non-parametric U test between the HI-insulted and control groups (Supplementary Table S4). The time effect was determined using either an ordinary one-way ANOVA test (Supplementary Table S7) or a non-parametric Kruskal-Wallis test (Supplementary Table S8) and multiple comparison analysis testing. The significance level was set at p < 0.05, and all p-values were adjusted for multiple testing using the Bonferroni method. Individual rats in the HI-insulted group were compared with prediction intervals of expected values based on the control group ( Supplementary Figs S17-S23).

Data availability. All data generated or analysed in this study are included in this published article and its supplementary information files.

Conclusions

In the present study, we simulated human neonatal HIE using a rat model and focused on the molecular and cellular processes in neonatal rat brains affected by HI insult. Acute stage cytotoxic oedema occurred as a result of cell swelling due to an influx of Na + , Cl − and Ca 2+ , followed by water entry into intracellular compartments from the interstitial space. In contrast, the subacute stage vasogenic oedema, representing an extracellular type of oedema related to blood-brain barrier disruption, resulted from plasma protein extravasation, followed by water accumulation within the interstitial space of the brain 34 . We followed these two distinct processes using a multimodal imaging approach. Importantly, the elemental MSI detected a low Na + abundance and high K + abundance in the control brain slices, reflecting the normal intracellular distribution of these cations 35 . The acute response to HI insult involved a significant increase in the concentration of elemental Na + and decrease in K + , which mirrored cell membrane depolarization. These respective alterations directly correlated with the observed changes in the profile of the Na + and K + adducts of the PCs as detected by MALDI MSI.

Based on the massive increase in NAPEs during the subacute phase, we speculate that their accumulation was the result of increased NAPE formation due to the degradation of the PI, PS and PA lipid species, whose intensities were decreased in the brain areas affected by HI injury. NAPE synthesis has been shown to increase under cellular stress and/or tissue damage and, as precursors of the N-acylethanolamine endocannabinoid ligands (NAE), NAPE may be involved in the endogenous neuroprotective reaction of the brain starting at the moment of an injurious process 36 . This finding correlates well with our results in which the accumulation of NAPEs started during the acute stage and then strongly increased due to progressive neurodegeneration during the subacute stage after the HI insult. Similarly to NAPE, we detected significantly higher intensities of GM2 and GM3 gangliosides in the affected brain regions compared to the non-affected areas and control brains. GM2 has been reported to accumulate along with the progressive destruction of neurons in a variety of neurological disorders 24 , including stroke and traumatic brain injuries 37,38 . The increased GM2/GM3 level may hence be considered as a marker of HI brain damage. Whether the observation of GM2 and GM3 just reflect injurious process, or these molecules are directly involved in the process of neuroprotection after the HI insult, both hypotheses still remain to be investigated 37,39 . To the best of our knowledge, there is just a single study that has reported alterations in brain gangliosides while using the same 7-day old rat experimental model of neonatal brain HI insult 40 . In that work an increased ganglioside GM3 level has been found in hippocampus of the exposed rat at 30 min and 1, 2 and 4 days after the HI insult. It is however difficult to compare those data with our findings due to substantially different tissue processing in the two studies. In the previous study 40 , an ex vivo processing of brain samples to get hippocampal homogenates, followed by laborious in vitro procedure and tissue extract analysis have been applied on the two hippocampal samples. In our study we applied in vivo fixation of the brain followed by in situ analysis of the entire lipidome in the whole brain section using mass spectrometry imaging techniques. Histopathological examination performed by us confirmed the significant impairment of the neuronal tissue, particularly in the cortical and striatum regions, which manifested as extensive liquefactive necrosis. Importantly, the most affected areas matched well with those of significantly increased NAPE, GM2 and GM3. Such increase of the particular lipid metabolites may reflect activation of neuroprotective mechanisms in the HI-insulted brain to minimize the damage.

Intriguingly, on the sixth day of HI injury, we observed a tendency of the neonatal rat brain to recover. We observed the disappearance of histopathological signs, and relatively intact neurons started to appear. These cells possessed morphological features that actually distinguished them from the control non-affected neurons. The concentration levels of neurodegenerative markers, such as NAPEs and GM2, decreased to nearly control levels, but a moderately increased level of GM3 was observed. According to the in vivo MR spectroscopy analysis of remarkably pronounced brain lesions in certain rats during the subacute stage of HI injury, animals that were severely affected by the insult are likely to suffer from adverse neurological outcomes. However, rats with apparently decreased brain lesions during the subacute stage subsequently showed recovery of the brain tissue. Overall, our findings are consistent with the fundamentals of current knowledge regarding this pathology, particularly its molecular and cellular processes, and should be considered in the development of novel therapeutic strategies for effective interventions in newborns affected by neonatal hypoxic-ischaemic encephalopathy. Along with modulating neurotrophin action by exogenous ganglioside analogues 41 , another way of neuroprotection and functional recovery of the damaged brain could involve (i) induction of endogenous NAPE formation and (ii) stimulation of NAPE transformation into neuroprotective NAEs, including those modulating CB receptors.

Figure 1 .
1Effects during the acute and subacute stages of HI insult. (A) -insulted pups curved to the left (#3 and 4), while the sham pups did not (#6). (B) -cytotoxic oedema development in the ipsilateral hemisphere (dotted line) and contralateral hemisphere (bold line) was detected immediately after HI insult at the striatal level (pup #16). (C) -3D image of the brain depicting the extent of the lesion (in red) in the ipsilateral hemisphere (pup #7); D-V and A-P black arrows indicate the dorso-ventral and anterior-posterior directions, respectively. (D) -subacute stage: areas of simultaneously occurring vasogenic and cytotoxic oedema are shown in green and red, respectively (pup #9). ScIentIfIc RePoRtS | (2018) 8:6952 | DOI:10.1038/s41598-018-25088-2

Figure 2 .
2Histological and MSI assessment of the HI-insulted neonatal rat brains during the acute stage of HIE. Panel A: Haematoxylin-eosin stained section demonstrating the early cellular response; a -expanded region of the ipsilateral hemisphere (indicated by the black rectangle shown in (A)) depicting the cytotoxic oedema-afflicted neurons with signs of swelling and vacuolization (red circles). MALDI MS images showing the altered distribution of PC (16:0/16:0) in the sodiated (panel B, m/z 756.550) and potassiated (panel D, m/z 772.525; signal was overscaled to 120% to provide better contrast) form. Quantitative LA-ICP-MS images of the elemental sodium (panel C) and potassium (panel E) distribution (for details see Supplementary Figure S4). MCtx (motor cortex), cc (corpus callosum), SCtx (somatosensory cortex), ec (external capsule), Cpu (caudate putamen = striatum). Different image scaling (B/D versus C/E) are due to different FlexImaging (Bruker Daltonics, Bremen, Germany) and ImageLab 42 programs. ScIentIfIc RePoRtS | (2018) 8:6952 | DOI:10.1038/s41598-018-25088-2 performing a parametric two-tailed t-test, with a p-value equal to 0.016 (Supplementary

Figure 3 .
3Subacute response to HI insult in the neonatal rat brain. (A) Haematoxylin-eosin stained section showing the ischaemic core of the lesion containing large-bodied astrocytes with an eosinophilic cytoplasm (cells indicated by black circles in the both expanded areas) and necrotic neuronal debris (red arrows, panel a); penumbra area (panel b) showing necrotic (white circles) and shapeless (black arrows) cells and intact neurons (green circle). MALDI MS images showing the altered distribution of NAPE (54:4) and GM2 (d18:1/18:0) in the affected area detected as deprotonated molecules at m/z 1004.768 (B) and 1382.805 (C), respectively. ScIentIfIc RePoRtS | (2018) 8:6952 | DOI:10.1038/s41598-018-25088-2 observed morphological changes, the neuronal cells had intact cellular membranes and preserved endoplasmic reticula (Supplementary

Figure 4 .
4Subacute alterations in the phospholipid profile in the HI-affected area of the neonatal rat brain. Illustrative spectra of the entire mass range acquired by negative ion MALDI from the ischaemic core (panel B) and the corresponding area in the sham control rat brain (panel A). Detailed arrays of NAPE and monosialoganglioside molecules are depicted in panels C and D, respectively.ScIentIfIc RePoRtS | (2018) 8:6952 | DOI:10.1038/s41598-018-25088-2 Materials and Methods Chemicals and reagents. The isoflurane anaesthetic and nitrogen 4.0 were obtained from Baxter and Linde (both Bratislava, Slovak Republic), respectively. The indium-tin oxide (ITO) glass slides and α-cyano-4-hydroxycinnamic acid (CHCA) were obtained from Bruker Daltonics (Bremen, Germany). Acetonitrile, methanol, ethanol and water (all LC-MS grade) were obtained from Merck Millipore (Prague, Czech Republic). Trifluoroacetic acid (TFA), chloroform, sodium chloride, 9-aminoacridine (9-AA), haematoxylin solution, Gill no. 2 and DPX mounting media were obtained from Sigma-Aldrich (Prague, Czech Republic). Eosin Y (0.5%) aqueous solution was purchased from VWR Chemicals (Stribrna Skalice, Czech Republic). Hydrochloric and nitric acid (65%, Analpure) were obtained from FlukaBioChemika or Analytika Ltd., respectively (both Prague, Czech Republic). Deionized water (18.2 MΩ/cm) was prepared using a Milli-Q system (Millipore, Molsheim, France). Omni Slides with hydrophobic surfaces were purchased from Prosolia, Inc. (Zionsville, USA). Model of hypoxic-ischaemic brain injury in rat neonates. All animal experiments were conducted in accordance with the EU legislation (Directive 2010/63/EU), regulations and guidelines of the Slovak Animal Protection Act (Directive No. 377/2012 and Regulation No. 436/2012) under the approval of the State Veterinary and Food Administration of the Slovak Republic (Decision No. 2575/11-221/3) and the Animal Welfare Committee of the Institute of Experimental Pharmacology and Toxicology, CEM of the Slovak Academy of Sciences in Bratislava.

. In the MALDI MSI, LA-ICP-MSI and histology analyses, consecutive sections of 10-, 30-and 30-µm

Figure 5 .
5Relative abundance of the NAPE (54:4), GM2, and GM3 species in the sham (blue) and HI-affected (red) rat neonatal brains at 0 hour, 36 hours and 144 hours after the HI insult. Statistical significance of the measurement is displayed at the *p < 0.05 and **p < 0.01 levels.ScIentIfIc RePoRtS | (2018) 8:6952 | DOI:10.1038/s41598-018-25088-2

and sham brains. During the early response of the brain to the HI insult, the intensities of the PCs at m/z 728.520, 754.536, 756.551, 782.567, and 784.583 increased, while the intensities at m/z 744.494, 770.510, 772.525, 798.541 and 800.557 decreased (SupplementaryTable S2). Individual species were defined by performing a CycloBranch exact mass search 18 against the LipidMaps database. Compared with the control brains, the sodium adducts of the PCs were increased, while the potassium adducts were decreased in theScIentIfIc RePoRtS | (2018) 8:6952 | DOI:10.1038/s41598-018-25088-2

-and 144-hour time points are shown in Supplementary Table S13. The results of the normality testing were nonhomogeneous; thus, both parametric and ScIentIfIc RePoRtS | (2018) 8:6952 | DOI:10.1038/s41598-018-25088-2
ScIentIfIc RePoRtS | (2018) 8:6952 | DOI:10.1038/s41598-018-25088-2
© The Author(s) 2018
AcknowledgementsThe authors gratefully acknowledge the help of Lukas Krasny (currently at the Institute of Cancer Research, London, UK), Kamila Salokyová (Faculty of Chemical and Food Technology, Bratislava, Slovakia), Martina Zíková (Institute of Molecular Genetics of the ASCR, v.v.i., Prague, Czech Republic) and Peter Makovický (Czech Centre for Phenogenomics, BIOCEV, Vestec, Czech Republic) during the early stages of the manuscript preparation. Support from the Ministry of Education, Youth and Sports of the Czech Republic (NPU LO1509), Czech Science Foundation (16-20229S) and VEGA 2/0155/16 (Slovakia) is also acknowledged.Author ContributionsThis manuscript was written with contributions from all authors. D.L. designed the experiments, handled the animals, and performed the sectioning, histology and MSI experiments; T.P. was responsible for ICP-MS; L.B. generated and evaluated the MRI data; B.S. performed the statistical evaluation; and D.L., A.S., I.J. and V.H. wrote the paper. All authors reviewed the manuscript.Additional InformationSupplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-25088-2.Competing Interests: The authors declare no competing interests.Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Neonatal brain injury as a consequence of insufficient cerebral oxygenation. K Placha, D Luptakova, L Baciak, E Ujhazy, I Juranek, Neuroendocrinol. Lett. 37Placha, K., Luptakova, D., Baciak, L., Ujhazy, E. & Juranek, I. Neonatal brain injury as a consequence of insufficient cerebral oxygenation. Neuroendocrinol. Lett. 37, 79-96 (2016).

Hypoxic-Ischemic Encephalopathy A Review for the Clinician. M Douglas-Escobar, M D Weiss, Jama Pediatrics. 169Douglas-Escobar, M. & Weiss, M. D. Hypoxic-Ischemic Encephalopathy A Review for the Clinician. Jama Pediatrics 169, 397-403 (2015).

Perinatal Hypoxic-Ischemic Encephalopathy. M C Lai, S N Yang, J. Biomed. Biotechnol. Lai, M. C. & Yang, S. N. Perinatal Hypoxic-Ischemic Encephalopathy. J. Biomed. Biotechnol (2011).

Fetal Hypoxia Insults and Patterns of Brain Injury: Insights from Animal Models. A J Gunn, L Bennet, Clin. Perinatol. 36Gunn, A. J. & Bennet, L. Fetal Hypoxia Insults and Patterns of Brain Injury: Insights from Animal Models. Clin. Perinatol. 36, 579-593 (2009).

Perinatal brain damage: The term infant. H Hagberg, A D Edwards, F Groenendaal, Neurobiol. Dis. 92Hagberg, H., Edwards, A. D. & Groenendaal, F. Perinatal brain damage: The term infant. Neurobiol. Dis. 92, 102-112 (2016).

Pathogenesis of hypoxic-ischemic brain injury. J M Perlman, J. Perinatol. 27Perlman, J. M. Pathogenesis of hypoxic-ischemic brain injury. J. Perinatol. 27, S39-S46 (2007).

Hypoxic-ischemic brain injury in the term infant-current concepts. L Shalak, J M Perlman, Early Hum. Dev. 80Shalak, L. & Perlman, J. M. Hypoxic-ischemic brain injury in the term infant-current concepts. Early Hum. Dev. 80, 125-141 (2004).

Progesterone attenuates cerebral edema in neonatal rats with hypoxic-ischemic brain damage by inhibiting the expression of matrix metalloproteinase-9 and aquaporin-4. X Y Wang, Exp. Ther. Med. 6Wang, X. Y. et al. Progesterone attenuates cerebral edema in neonatal rats with hypoxic-ischemic brain damage by inhibiting the expression of matrix metalloproteinase-9 and aquaporin-4. Exp. Ther. Med. 6, 263-267 (2013).

Hypoxic-ischemic injury in the developing brain: the role of reactive oxygen species originating in mitochondria. V S Ten, A Starkov, Neurol. Res. Int. 2012542976Ten, V. S. & Starkov, A. Hypoxic-ischemic injury in the developing brain: the role of reactive oxygen species originating in mitochondria. Neurol. Res. Int. 2012, 542976 (2012).

Molecular Mechanisms of NeonatalBrain Injury. C Thornton, Neurol. Res. Int. 201216Thornton, C. et al. Molecular Mechanisms of NeonatalBrain Injury. Neurol. Res. Int. 2012, 16 (2012).

The mechanisms and treatment of asphyxial encephalopathy. G Wassink, E R Gunn, P P Drury, L Bennet, A J Gunn, Front. Neurosci. 840Wassink, G., Gunn, E. R., Drury, P. P., Bennet, L. & Gunn, A. J. The mechanisms and treatment of asphyxial encephalopathy. Front. Neurosci. 8, 40 (2014).

Roles of fatty acid ethanolamides (FAE) in traumatic and ischemic brain injury. E Esposito, M Cordaro, S Cuzzocrea, Pharmacol. Res. 86Esposito, E., Cordaro, M. & Cuzzocrea, S. Roles of fatty acid ethanolamides (FAE) in traumatic and ischemic brain injury. Pharmacol. Res. 86, 26-31 (2014).

Therapeutic hypothermia for newborn infants with hypoxic-ischaemic encephalopathy. S E Jacobs, W O Tarnow-Mordi, J. Paediatr. Child Health. 46Jacobs, S. E. & Tarnow-Mordi, W. O. Therapeutic hypothermia for newborn infants with hypoxic-ischaemic encephalopathy. J. Paediatr. Child Health 46, 568-576 (2010).

Moderate Hypothermia to Treat Perinatal Asphyxial Encephalopathy. D V Azzopardi, N. Engl. J. Med. 361Azzopardi, D. V. et al. Moderate Hypothermia to Treat Perinatal Asphyxial Encephalopathy. N. Engl. J. Med. 361, 1349-1358 (2009).

Perinatal asphyxia in the term newborn. R Antonucci, A Porcella, M D Pilloni, J. Pediat. Neonat. Individual. Med. 3Antonucci, R., Porcella, A. & Pilloni, M. D. Perinatal asphyxia in the term newborn. J. Pediat. Neonat. Individual. Med. 3, 1-14 (2014).

Cerebral Palsy after Neonatal Encephalopathy: How Much Is Preventable?. J Garfinkle, J. Pediatr. 167Garfinkle, J. et al. Cerebral Palsy after Neonatal Encephalopathy: How Much Is Preventable? J. Pediatr. 167, 58-U417 (2015).

Hypothermia to treat neonatal hypoxic ischemic encephalopathy: systematic review. P S Shah, A Ohlsson, M Perlman, Arch. Pediatr. Adolesc. Med. 161Shah, P. S., Ohlsson, A. & Perlman, M. Hypothermia to treat neonatal hypoxic ischemic encephalopathy: systematic review. Arch. Pediatr. Adolesc. Med. 161, 951-958 (2007).

Batch-processing of Imaging or Liquid-Chromatography Mass Spectrometry Datasets and De Novo Sequencing of Polyketide Siderophores. J Novak, BBA Prot. Proteom. 1865Novak, J. et al. Batch-processing of Imaging or Liquid-Chromatography Mass Spectrometry Datasets and De Novo Sequencing of Polyketide Siderophores. BBA Prot. Proteom. 1865, 768-775 (2017).

Age dependent accumulation of N-acyl-ethanolamine phospholipids in ischemic rat brain. A (31)P NMR and enzyme activity study. B Moesgaard, G Petersen, J W Jaroszewski, H S Hansen, J. Lipid Res. 41Moesgaard, B., Petersen, G., Jaroszewski, J. W. & Hansen, H. S. Age dependent accumulation of N-acyl-ethanolamine phospholipids in ischemic rat brain. A (31)P NMR and enzyme activity study. J. Lipid Res. 41, 985-990 (2000).

Massive accumulation of N-acylethanolamines after stroke. Cell signalling in acute cerebral ischemia?. C Berger, J. Neurochem. 88Berger, C. et al. Massive accumulation of N-acylethanolamines after stroke. Cell signalling in acute cerebral ischemia? J. Neurochem. 88, 1159-1167 (2004).

Changes in brain levels of N-acylethanolamines and 2-arachidonoylglycerol in focal cerebral ischemia in mice. M Degn, J. Neurochem. 103Degn, M. et al. Changes in brain levels of N-acylethanolamines and 2-arachidonoylglycerol in focal cerebral ischemia in mice. J. Neurochem. 103, 1907-1916 (2007).

Increased severity of stroke in CB1 cannabinoid receptor knock-out mice. S Parmentier-Batteur, K Jin, X O Mao, L Xie, D Greenberg, J. Neurosci. 22Parmentier-Batteur, S., Jin, K., Mao, X. O., Xie, L. & Greenberg, D. A. Increased severity of stroke in CB1 cannabinoid receptor knock-out mice. J. Neurosci. 22, 9771-9775 (2002).

Changes in N-acylethanolamine Pathway Related Metabolites in a Rat Model of Cerebral Ischemia/Reperfusion. A Kilaru, J. Glyc. Lipidomics. 1Kilaru, A. et al. Changes in N-acylethanolamine Pathway Related Metabolites in a Rat Model of Cerebral Ischemia/Reperfusion. J. Glyc. Lipidomics 1 (2011).

Biochemical consequences of mutations causing the GM2 gangliosidoses. D J Mahuran, Biochim. Biophys. Acta. 1455Mahuran, D. J. Biochemical consequences of mutations causing the GM2 gangliosidoses. Biochim. Biophys. Acta 1455, 105-138 (1999).

Secondary energy failure after cerebral hypoxia-ischemia in the immature rat. R C Vannucci, J Towfighi, S J Vannucci, J. Cereb. Blood Flow Metab. 24Vannucci, R. C., Towfighi, J. & Vannucci, S. J. Secondary energy failure after cerebral hypoxia-ischemia in the immature rat. J. Cereb. Blood Flow Metab. 24, 1090-1097 (2004).

The influence of immaturity on hypoxic-ischemic brain-damage in the rat. J E Rice, R C Vannucci, J B Brierley, Ann. Neurol. 9Rice, J. E., Vannucci, R. C. & Brierley, J. B. The influence of immaturity on hypoxic-ischemic brain-damage in the rat. Ann. Neurol. 9, 131-141 (1981).

Longitudinal MR Assessment of Hypoxic Ischemic Injury in the Immature Rat Brain. Y Van De Looij, A Chatagner, P S Huppi, R Gruetter, S V Sizonenko, Magn. Reson. Med. 65van de Looij, Y., Chatagner, A., Huppi, P. S., Gruetter, R. & Sizonenko, S. V. Longitudinal MR Assessment of Hypoxic Ischemic Injury in the Immature Rat Brain. Magn. Reson. Med. 65, 305-312 (2011).

Atlas of the Developing Rat Brain. N Sherwood, P S Timiras, Stereotaxic, University of California PressSherwood, N. & Timiras, P. S. A Stereotaxic Atlas of the Developing Rat Brain (University of California Press, 1970).

De Novo Sequencing of Nonribosomal Peptides from Accurate Product Ion Mass Spectra. J Novak, K Lemr, K A Schug, V Havlicek, Cyclobranch, J. Am. Soc. Mass Spectrom. 26Novak, J., Lemr, K., Schug, K. A. & Havlicek, V. CycloBranch: De Novo Sequencing of Nonribosomal Peptides from Accurate Product Ion Mass Spectra. J. Am. Soc. Mass Spectrom. 26, 1780-1786 (2015).

Chemometric Analysis of Multisensor Hyperspectral Images of Precipitated Atmospheric Particulate Matter. J Ofner, Anal. Chem. 87Ofner, J. et al. Chemometric Analysis of Multisensor Hyperspectral Images of Precipitated Atmospheric Particulate Matter. Anal. Chem. 87, 9413-9420 (2015).

Application of dried-droplets deposited on pre-cut filter paper disks for quantitative LA-ICP-MS imaging of biologically relevant minor and trace elements in tissue samples. M Bonta, B Hegedus, A Limbeck, Anal. Chim. Acta. 908Bonta, M., Hegedus, B. & Limbeck, A. Application of dried-droplets deposited on pre-cut filter paper disks for quantitative LA-ICP- MS imaging of biologically relevant minor and trace elements in tissue samples. Anal. Chim. Acta 908, 54-62 (2016).

Radial line-scans as representative sampling strategy in dried-droplet laser ablation of liquid samples deposited on pre-cut filter paper disks. W Nischkauer, F Vanhaecke, S Bernacchi, C Hervvig, A Limbeck, Spectrochim. Acta Part B-Atomic Spec. 101Nischkauer, W., Vanhaecke, F., Bernacchi, S., Hervvig, C. & Limbeck, A. Radial line-scans as representative sampling strategy in dried-droplet laser ablation of liquid samples deposited on pre-cut filter paper disks. Spectrochim. Acta Part B-Atomic Spec. 101, 123-129 (2014).

NIH Image to ImageJ: 25 years of image analysis. C A Schneider, W S Rasband, K W Eliceiri, Nature Methods. 9Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nature Methods 9, 671-675 (2012).

Molecular pathophysiology of cerebral edema. J A Stokum, V Gerzanich, J M Simard, J. Cereb. Blood Flow Metab. 36Stokum, J. A., Gerzanich, V. & Simard, J. M. Molecular pathophysiology of cerebral edema. J. Cereb. Blood Flow Metab. 36, 513-538 (2016).

Role of Phosphatidylcholine During Neuronal Differentiation. L Paoletti, C Elena, P Domizi, C Banchio, Iubmb Life. 63Paoletti, L., Elena, C., Domizi, P. & Banchio, C. Role of Phosphatidylcholine During Neuronal Differentiation. Iubmb Life 63, 714-720 (2011).

biosynthesis and functions of N-acylphosphatidylethanolamines (NAPE): not just precursors of N-acylethanolamines (NAE). D Coulon, L Faure, M Salmon, V Wattelet, J J Bessoule, Occurrence, Biochimie. 94Coulon, D., Faure, L., Salmon, M., Wattelet, V. & Bessoule, J. J. Occurrence, biosynthesis and functions of N-acylphosphatidylethanolamines (NAPE): not just precursors of N-acylethanolamines (NAE). Biochimie 94, 75-85 (2012).

Mass spectrometry imaging of biomarker lipids for phagocytosis and signalling during focal cerebral ischaemia. M M Nielsen, Sci. Rep. 639571Nielsen, M. M. et al. Mass spectrometry imaging of biomarker lipids for phagocytosis and signalling during focal cerebral ischaemia. Sci. Rep. 6, 39571 (2016).

Imaging mass spectrometry detection of gangliosides species in the mouse brain following transient focal cerebral ischemia and long-term recovery. S N Whitehead, PLoS One. 620808Whitehead, S. N. et al. Imaging mass spectrometry detection of gangliosides species in the mouse brain following transient focal cerebral ischemia and long-term recovery. PLoS One 6, e20808 (2011).

Monosialoganglioside GM1 treatment after a hypoxic-ischemic episode reduces the vulnerability of the fetal sheep brain to subsequent injuries. W K M Tan, C E Williams, C E Mallard, P D Gluckman, Am. J. Obstet. Gynecol. 170Tan, W. K. M., Williams, C. E., Mallard, C. E. & Gluckman, P. D. Monosialoganglioside GM1 treatment after a hypoxic-ischemic episode reduces the vulnerability of the fetal sheep brain to subsequent injuries. Am. J. Obstet. Gynecol. 170, 663-670 (1994).

Effects of Neonatal Hypoxia/Ischemia on Ganglioside Expression in the Rat Hippocampus. V M T Trindade, Neurochem. Res. 26Trindade, V. M. T. et al. Effects of Neonatal Hypoxia/Ischemia on Ganglioside Expression in the Rat Hippocampus. Neurochem. Res. 26, 591-597 (2001).

Targeting Neurotrophin Receptors in the Central Nervous System. I Mocchetti, M Brown, CNS & Neurol. Dis. -Drug Targets. 7Mocchetti, I. & Brown, M. Targeting Neurotrophin Receptors in the Central Nervous System. CNS & Neurol. Dis. -Drug Targets 7, 71-82 (2008).

Multisensor hyperspectral imaging as a versatile tool for image-based chemical structure determination. H Lohninger, J Offer, Spectrosc. Eur. 26Lohninger, H. & Offer, J. Multisensor hyperspectral imaging as a versatile tool for image-based chemical structure determination. Spectrosc. Eur. 26, 6-10 (2014).


Milk glucosidase activity enables suckled pup starch digestion

B L Nichols
Children's Nutrition Research Center, Baylor College of Medicine and Texas Children's Hospital
HoustonTXUSA

M Diaz-Sotomayor
Children's Nutrition Research Center, Baylor College of Medicine and Texas Children's Hospital
HoustonTXUSA

S E Avery
Children's Nutrition Research Center, Baylor College of Medicine and Texas Children's Hospital
HoustonTXUSA

S K Chacko
Children's Nutrition Research Center, Baylor College of Medicine and Texas Children's Hospital
HoustonTXUSA

D L Hadsell
Children's Nutrition Research Center, Baylor College of Medicine and Texas Children's Hospital
HoustonTXUSA

S S Baker
B R Hamaker
L K Yan
H M Lin
R Quezada-Calvillo
Children's Nutrition Research Center, Baylor College of Medicine and Texas Children's Hospital
HoustonTXUSA

M I N I R E V I E W

Milk glucosidase activity enables suckled pup starch digestion
10.1186/s40348-016-0032-zWeaningSucklingStarch digestionMaltase-glucoamylaseSucrase-isomaltaseGlucogenesis from 13 C-starch
Starch requires six enzymes for digestion to free glucose: two amylases (salivary and pancreatic) and four mucosal maltase activities; sucrase-isomaltase and maltase-glucoamylase. All are deficient in suckling rodents. Objective: The objective of this study is to test 13 C-starch digestion before weaning by measuring enrichment of blood 13 C-glucose in maltase-glucoamylase-null and wild-type mice. Methods: Maltase-glucoamylase gene was ablated at the N-terminal. Dams were fed low 13 C-diet and litters kept on low 13 C-diet. Pups were weaned at 21 days. Digestion was tested at 13 and 25 days by intragastric feeding of amylase predigested 13 C-α-limit dextrins. Blood 13 C-glucose enrichment was measured by gas chromatography combustion isotope ratio mass spectrometry (GCRMS) using penta-acetate derivatives. Results: Four hours after feeding, blood 13 C-glucose was enriched by 26 × 10 3 in null and 18 × 10 3 in wild-type mice at 13 days and 0.3 × 10 3 and 0.2 × 103 at 25 days (vs. fasting p = 0.045 and p = 0.045). By jejunal enzyme assay, immunohistochemistry, or Western blots, there was no maltase activity or brush border staining with maltaseglucoamylase antibodies at 13 days, but these were fully developed in the wild-type mice by 25 days. In 13-day null mice, luminal contents were stained by maltase-glucoamylase antibodies. Lactating the mammary gland revealed maltase-glucoamylase antibody staining of alveolar cells. Reverse transcription/polymerase chain reaction (RT/PCR) of lactating glands revealed a secreted form of maltase-glucoamylase. Conclusions: (1) 13 C-α-limit dextrins were rapidly digested to 13 C-glucose in 13-day mice independent of maltaseglucoamylase genotype or mucosal maltase activity. (2) This experiment demonstrates that a soluble maltase activity is secreted in mouse mother's milk which enables suckling pup starch digestion well before brush border enzyme development. (3) This experiment with 13 C-α-limit dextrins needs to be repeated in human breast fed infants.

The word "mammal" is from the scientific name Mammalia derived from the Latin mamma ("teat"). All female mammals suckle their young with milk which is secreted from apocrine mammary glands. Only the young are fed mother's milk, and the process of suckling cessation is called weaning. For human infants, supplementation of maternal nursing is called complementary feeding or beikost [1]. The German word beikost translates as "infant foods other than milk or formula." Starchy foods are the first offered as beikost in most cultures [2]. The introduction of beikost begins the weaning process and introduces environmental vectors stressing the maternalinfant dyad. The presently recommended age for introduction of complementary feedings to the suckling infant is 6 months [2]. One of the arguments for this controversial recommendation offered by learned committees is the physiologic delay of amylase production and secretion in normal infants. Pediatricians worldwide know that many mothers start offering beikost in the first months of life and that this common practice is usually rewarded by infant behavior. On the other hand, it is believed that in developing counties, much of the stunting of infant growth is due to poor nutritional quality and environmental contamination of complementary feedings [3]. With a general hypothesis that the starch offered in complementary foods is nutritionally available to in the suckling infant, we designed the following set of experiments in a mouse model. As in the human infant, developmental amylase insufficiency is present in the mouse. In suckling rodents, amylase and mucosal glucoamylase insufficiency are well documented [4]. This led to our experimental hypothesis that the suckling mouse can digest starch before the age of formal weaning, 21 days. The maltase-glucoamylase (Mgam) gene was ablated at the N-terminal expressing the membrane binding domain [5], and this hypothesis was tested in mucosal Mgam knockout (KO) and wildtype (WT) mice before and after weaning. In these experiments, it was shown that suckling mice begin to consume the adult diet by 15 days and that they can digest food starch before that age. The source of the starch digesting glucosidase activity was shown to be maternal milk which is expressed from the lactating alveolar cells of the mammary gland in a smaller molecular weight soluble form when compared to the membrane-bound form of the wild-type adult mouse jejunum.

Results and discussion

Mgam KO on in vitro suckling starch digestion

Methods: Genotyped mice were bred to produce Mgam KO and WT pups [5,6]. Dates of birth were closely monitored for accuracy of ages. All the suckling mice had clotted milk visible in stomach at time of sacrifice. One-centimeter segments were removed from the midjejunum and conserved at −70°C for subsequent glucosidase assays [6]. After flushing the lumen, the tissue was homogenized in phosphate-buffered saline (PBS) buffer with ethylene diamine tetra-acetic acid (EDTA). Aliquots of the homogenate and flush were assayed for glucose production from maltose substrate by the Dahlqvist glucose-oxidase method. Results: After weaning at 21 days, there was a clear difference between null and WT genotype ( Fig. 1) jejunum homogenate αglucosidase activities at 13 and 90 days of age in eight null and eight WT mice. Conclusion: There was confirmation that mucosal-bound Mgam was ablated in weaned KO mice and that all α-glucosidase activities were absent in both null and WT suckling mice.

In vivo digestion of starch in suckling mice

Methods: To test starch digestion, we used amylasepretreated UL 13 C-starch limit dextrin ( 13 C-LDx) [7] which was fed by gavage to 13-day-old suckling mice with either KO or WT Mgam genotypes. These same genotypes were tested again at 25 days, 4 days after weaning. Mating of four to six females fed low 13 C-diet (TD.06089, based upon rice starch) for each mouse genotype, enough to produce four litters of the same (±6 h) age for each. The resulting litters were kept under low 13 C-feeding (lactating) and housing [8]. Twenty-four mice were required for the study. At 21 days, the mice were weaned in individual cages to low 13 C-diet with feeding permitted 24 h. Four mice were taken from the null and four from the WT genotypes, and blood and tissue were collected 4 h after intragastric feeding of 13 C-LDx. Trunk blood was collected in fluoride/EDTA tubes at 4°C and spun and frozen at 70°C until analysis. Enrichments of plasma 13 C-glucose (starch glucogenesis) were measured by gas chromatography combustion isotope ratio mass spectrometry (GCRMS) using the pentaacetate derivative. Jejunal α-glucosidase activities were measured as above. Results: The mucosal α-glucosidase phenotypes confirmed the genotypes of the weaned mice. Four hours after 13 C-LDx feeding, blood 13 C-glucose was enriched by 26 × 10 3 in null and 18 × 10 3 in WT at 13 days and 0.3 × 10 3 and 0.2 × 103 at 25 days (vs. fasting p = 0.045 and p = 0.045). Conclusion: Fed 13 C-LDx was rapidly digested and transported to enrich blood 13 C-glucose in 13-and 25-day mice independent of Mgam genotype or jejunal homogenate phenotype.

C-terminal Mgam epitope in suckling jejunum lumen

Methods: Recombinant mouse C-terminal Mgam was used to immunize rabbits [9]. The resulting polyclonal antibody (pAb) was specific as evidenced by blocking with the immunizing peptide and absence of reaction with C-terminal sucrase-isomaltase (Si) immunoprecipitates from the mouse mucosa. Results: Western blots of whole jejunal homogenates of triplicate null and WT 13day suckling mice revealed three bands at 60-120 KDa that were also visualized at 3 months, but the mature pattern of five bands at 130-400 was only seen in the weaned WT homogenates (Fig. 2). Immunohistology of jejunum revealed staining of luminal milk in both null and WT 13-day mice but only staining of the brush border in the 3-month WT jejunum. Conclusion: A specific rabbit antibody against mouse Mgam recognizes a smaller MW epitope in suckling mice which is visualized as milk residue in the lumen of the jejunum. The higher MW Western blot bands and jejunal brush border staining patterns are only visualized in the weaned WT mice.

C-terminal Mgam epitope in mouse milk and lactating gland

Method: Mammary tissue was removed from a WT lactating mouse at 20 days post-partum. Milk was obtained by vacuum milking from three 20-day post-partum lactating dams and frozen for Western blotting. By immunohistology, the lactating gland was diffusely stained with the Mgam pAb at low power, and staining of the milk-producing alveolar cells and ductal lumen contents was clearly visualized on higher power. The Western blot from three 20-day lactating C57BL/6J mouse milks revealed the expected higher MW Mgam bands from control WT mouse jejunum and only the lower MW bands similar to those of suckling mice in Fig. 2. Conclusion: The epitope for glucoamylase is visualized by immunohistology of alveolar cells of the mouse lactating mammary gland and is also recognized by Western blots of mouse milk. Fig. 1 Dahlqvist disaccharidase assays from the mid-jejunum of suckling and weaned mice with Mgam KO or WT genotypes (n = 8). Weaning was by separation from the litter at 21 days. Tissues were collected at 13 days (suckling) and 90 days (weaned). The lumen was first flushed with EDTA PBS buffer, and then, tissue was collected and homogenized in PBS buffer and analyzed as unit per milligram protein (a). The luminal flush (b) was assayed for activity per milliliter of collected fluid. Note that the homogenate activities for maltose, sucrose, and isomaltose substrates increased after weaning in the WT but not in the null mice. Note also the difference in ordinate scales between (a) and (b)

Secreted form of Mgam mRNA in lactating gland

Method: Lactating mammary glands were harvested from ten different strains of mice (129S1/SvImJ, C57BL/ 6J, CBA/J, BTBRT<+>tf/J, Qsi5, MA/MyJ. A/J, Sea/GnJ, FVB/NJ, and CZECHIII/Eij strains) at 20 days postpartum. The tissues were snap frozen and stored for assay. Method and results: WT mouse jejunal RNA served as positive control. RNA was extracted as previously described [10]. A first reverse transcription/polymerase chain reaction (RT/PCR) had 5′ end primers anchored in exon 1 for both Si (exon 1 to exon 6) and Mgam (exon 1 to exon 7), and only mouse jejunal RNA samples were amplified on the first RT/PCR. In a second RT/PCR, Si (exon 16 to exon 18) did not amplify from the mammary gland RNAs, but the N-terminal Mgam (exon 3 to exon 7) did amplify which suggests that exon 1 for Mgam is spliced out for lactating mammary gland [10]. Conclusion: RT/PCR of lactating glands from ten different mouse strains revealed a message for a secreted form of Mgam, lacking the membrane binding coding exon.

Weaning process in suckling mice

From an animal management perspective, mouse weaning took place at 21 days of life when the pups are separated into individual cages. From a nutritional perspective, the feeding on mouse chow was begun during suckling. In the Mgam-null mouse, the coexistence of stained milk globules and unstained undigested plant structures were evident by age 15 days. This is consistent with the term weaning which implies a gradual transition from maternal to external food sources.

Weaning

From a chemical perspective, there is a major shift in food carbohydrate substrate affinity during weaning. Lactose is the major disaccharide in mother's milk. It is linked by a β-linkage between glucose and galactose. The digestible dietary carbohydrates of foods are, with the exception of milk, all α-linked glucose oligomers, while the indigestible cell structures, usually distally fermented, are β-linked fibers. In this context, mammalian weaning is a developmental shift of small intestinal βto α-glucosidic substrate digestion, a shift from a carnivorous to an omnivorous diet in human infants.

The major limitation to this investigation was the necessary use of a mouse Mgam KO model [6]. In contrast to the suckling mouse, humans have full mucosal Mgam and Si activities in jejunal mucosa from before birth [11]. The physiologic delay in salivary and pancreatic amylase production and secretion [12,13] parallels the rodent [14] and other model species. In our in vivo experimental model in mice, we removed the question about amylase maturation from consideration by exhaustive pre-digestion the test starch with amylase to Fig. 2 Western blots of mid-jejunal homogenates from mice with Mgam KO or WT genotypes before (13 days) and after (90 days) weaning. Mgam KO and WT were analyzed at each age (n = 3). Molecular weight standards are run in the left two columns and a negative control, tissue from Mgam deficient adult CBA Ca strain mouse, in the right column. The rabbit anti-mouse Mgam polyclonal served as primary antibody (see text). Note that only the 90-day WT tissue had the characteristic bands associated with Mgam but that smaller bands were recognized in all mice. These smaller bands were also visualized on Western blots of mouse milk (see text) make limit dextrins (LDx) [8]. Mucosal activities were tested in vitro with maltose made by amylase digestion of starch [7]. Mucosal glucogenesis from LDx is commonly termed α-glucosidase activity although the same mucosal activity is also recognized with maltose substrate, the major product of starch hydrolysis by amylase. As assayed in vitro, mucosal activity is a composite of four different enzymes expressed from two genes, maltase-glucoamylase (Mgam) and sucrase-isomaltase (Si), each with an N-terminal and C-terminal maltase activity. There are differences in rates and substrate specificities of the four maltase activities [7]. The C-terminal of Mgam is the most active for maltose and long chain or branched oligosaccharide substrate glucogenesis. Mgam activities are almost independent of amylase hydrolysis, but Si activities are greatly amplified by amylase pre-hydrolysis of starches. This has led to the view that glucoamylase activities suffice for starch digestion during physiologic amylase insufficiency [15]. This set of experiments suggests that secreted enzymes in mother's milk participate in digestion of complementary starch in mice. Reported studies of human milk demonstrate the presence of amylase peptide and activity as part of the functional components in human milk [16][17][18][19]. Could the amylase activity secreted in mother's milk amplify the glucogenic activities present in the human infant's small intestine?

Pediatric nutritional implications

There is no direct proof that human infants can digest complementary food starches in the small intestine, but indirect evidence exists. Studies of starch feeding show that both whole cereal starch and various maltodextrins (MDx) are digested by the young infant [20,21]. Evidence from complementary feeding studies of nursing infants in affluent settings reveals that complementary foods amplify energy intakes and sustain growth [22]. However, in challenging environments, complementary feeding may fail to sustain infant growth [3]. The problem of infant stunting is of global significance [3,23,24]. While not addressing this global pediatric issue directly, this experiment shows that suckling starch digestion is enabled by mouse maternal nursing and suggests that human breast milk feeding may enable amplified starch digestion in the suckling infant. This hypothesis is testable by the minimally invasive 13 C-LDx feeding procedure used in experiment 2 of this mouse investigation.

 AcknowledgementsThis project was funded in part by the United States Department of Agriculture, Agricultural Research Service Cooperative Agreement 58+6250-1-003, at Baylor College of Medicine to BLN and AQC and by internal funds from the Whistler Center for Carbohydrate Research, Purdue University, to BRH.Competing interestsThe authors declare that they have no competing interests.Authors' contributions BLN, MD-S, BRH, and RQ-C developed the concept. SEA managed the mouse colony and performed PCR. MD-S and LKY participated in the animal experiments. SSB and RQ-C did disaccharidase assays. SKC did 13 C-glucose assays. DLH and LHN provided the essential test materials. BLN drafted this mini-review. All authors read and approved the final manuscript.
Infant feeding in the 20th century: formula and beikost. S Fomon, J Nutr. 1312Fomon S (2001) Infant feeding in the 20th century: formula and beikost. J Nutr 131(2):409S-420S

European Childhood Obesity Project (2010) Introduction of complementary feeding in 5 European countries. S Schiess, V Grote, S Scaglioni, V Luque, F Martin, A Stolarczyk, F Vecchi, B Koletzko, J Pediatr Gastroenterol Nutr. 501Schiess S, Grote V, Scaglioni S, Luque V, Martin F, Stolarczyk A, Vecchi F, Koletzko B, European Childhood Obesity Project (2010) Introduction of complementary feeding in 5 European countries. J Pediatr Gastroenterol Nutr 50(1):92-98

Systematic review of the efficacy and effectiveness of complementary feeding interventions in developing countries. K G Dewey, S Adu-Afarwuah, Matern Child Nutr. 41SupplDewey KG, Adu-Afarwuah S (2008) Systematic review of the efficacy and effectiveness of complementary feeding interventions in developing countries. Matern Child Nutr 4(Suppl 1):24-85

Intestinal glycosidase activities in the human embryo, fetus, and newborn. S Auricchio, A Rubino, G Muerset, Pediatrics. 35Auricchio S, Rubino A, Muerset G (1965) Intestinal glycosidase activities in the human embryo, fetus, and newborn. Pediatrics 35:944-954

Contribution of mucosal maltase-glucoamylase activities to mouse small intestinal starch alpha-glucogenesis. R Quezada-Calvillo, C C Robayo-Torres, A R Opekun, P Sen, Z Ao, B R Hamaker, A Quaroni, G D Brayer, S Wattler, M C Nehls, E E Sterchi, B L Nichols, J Nutr. 1377Quezada-Calvillo R, Robayo-Torres CC, Opekun AR, Sen P, Ao Z, Hamaker BR, Quaroni A, Brayer GD, Wattler S, Nehls MC, Sterchi EE, Nichols BL (2007) Contribution of mucosal maltase-glucoamylase activities to mouse small intestinal starch alpha-glucogenesis. J Nutr 137(7):1725-1733

Mucosal maltase-glucoamylase plays a crucial role in starch digestion and prandial glucose homeostasis of mice. B L Nichols, R Quezada-Calvillo, C C Robayo-Torres, Z Ao, B R Hamaker, N F Butte, J Marini, F Jahoor, E E Sterchi, J Nutr. 1394Nichols BL, Quezada-Calvillo R, Robayo-Torres CC, Ao Z, Hamaker BR, Butte NF, Marini J, Jahoor F, Sterchi EE (2009) Mucosal maltase-glucoamylase plays a crucial role in starch digestion and prandial glucose homeostasis of mice. J Nutr 139(4):684-690

Mucosal C-terminal maltase-glucoamylase hydrolyzes large size starch digestion products that may contribute to rapid postprandial glucose generation. B H Lee, A H Lin, B L Nichols, K Jones, D R Rose, R Quezada-Calvillo, B R Hamaker, Mol Nutr Food Res. 585Lee BH, Lin AH, Nichols BL, Jones K, Rose DR, Quezada-Calvillo R, Hamaker BR (2014) Mucosal C-terminal maltase-glucoamylase hydrolyzes large size starch digestion products that may contribute to rapid postprandial glucose generation. Mol Nutr Food Res 58(5):1111-1121

Maltase-glucoamylase modulates gluconeogenesis and sucrase-isomaltase dominates starch digestion glucogenesis. M Diaz-Sotomayor, R Quezada-Calvillo, S E Avery, S K Chacko, L K Yan, A H Lin, Z H Ao, B R Hamaker, B L Nichols, 10.1097/MPG.0b013e3182a27438J Pediatr Gastroenterol Nutr. 576PubMedDiaz-Sotomayor M, Quezada-Calvillo R, Avery SE, Chacko SK, Yan LK, Lin AH, Ao ZH, Hamaker BR, Nichols BL (2013) Maltase-glucoamylase modulates gluconeogenesis and sucrase-isomaltase dominates starch digestion glucogenesis. J Pediatr Gastroenterol Nutr 57(6):704-712. doi:10.1097/MPG. 0b013e3182a27438, PubMed

Enterocyte loss of polarity and gut wound healing rely upon the F-actin-severing function of villin. F Ubelmann, M Chamaillard, F El-Marjou, A Simon, J Netter, D Vignjevic, B L Nichols, R Quezada-Calvillo, T Grandjean, D Louvard, C Revenu, S Robine, 10.1073/pnas.121844611023520048PMC3625339Proc Natl Acad Sci U S A. 11015Ubelmann F, Chamaillard M, El-Marjou F, Simon A, Netter J, Vignjevic D, Nichols BL, Quezada-Calvillo R, Grandjean T, Louvard D, Revenu C, Robine S (2013) Enterocyte loss of polarity and gut wound healing rely upon the F-actin-severing function of villin. Proc Natl Acad Sci U S A 110(15):E1380-E1389. doi:10.1073/pnas.1218446110, PubMed PMID: 23520048, PubMed Central PMCID: PMC3625339, Epub 2013 Mar 21

Human small intestinal maltase-glucoamylase cDNA cloning. Homology to sucrase-isomaltase. B L Nichols, J Eldering, S Avery, D Hahn, A Quaroni, E Sterchi, J Biol Chem. 2735PubMedNichols BL, Eldering J, Avery S, Hahn D, Quaroni A, Sterchi E (1998) Human small intestinal maltase-glucoamylase cDNA cloning. Homology to sucrase-isomaltase. J Biol Chem 273(5):3076-3081, PubMed

Development of carbohydrate absorption in the fetus and neonate. M Mobassaleh, R K Montgomery, J A Biller, R J Grand, Pediatrics. 751Pt 2. PubMedMobassaleh M, Montgomery RK, Biller JA, Grand RJ (1985) Development of carbohydrate absorption in the fetus and neonate. Pediatrics 75(1 Pt 2):160-166, PubMed

Ontogeny of human pancreatic exocrine function. P Mcclean, L T Weaver, 7679890Arch Dis Child. 6811029173Review. PubMed. PubMed Central PMCIDMcClean P, Weaver LT. Ontogeny of human pancreatic exocrine function. Arch Dis Child. 1993; 68(1 Spec No):62-5. Review. PubMed PMID: 7679890; PubMed Central PMCID: PMC1029173.

Effect of early weaning and prolonged nursing on development of the rat pancreas. P C Lee, O K Kim, E Lebenthal, Pediatr Res. 166PubMedLee PC, Kim OK, Lebenthal E (1982) Effect of early weaning and prolonged nursing on development of the rat pancreas. Pediatr Res 16(6):470-473, PubMed

Developmental pattern of rat intestinal brush-border enzymic proteins along the villus-crypt axis. P M Simon, M Kedinger, Raul F Grenier, J F Haffen, K , 109086Biochem J. 17821186529Simon PM, Kedinger M, Raul F, Grenier JF, Haffen K (1979) Developmental pattern of rat intestinal brush-border enzymic proteins along the villus-crypt axis. Biochem J 178(2):407-413, PubMed PMID: 109086, PubMed Central PMCID: PMC1186529

Direct starch digestion by sucrase-isomaltase and maltase-glucoamylase. A H Lin, B R Hamaker, B L NicholsJr, J Pediatr Gastroenterol Nutr. 552SupplLin AH, Hamaker BR, Nichols BL Jr (2012) Direct starch digestion by sucrase-isomaltase and maltase-glucoamylase. J Pediatr Gastroenterol Nutr 55(Suppl 2):S43-S45

Nutritional and physiologic significance of human milk proteins. B Lönnerdal, Am J Clin Nutr. 776Lönnerdal B (2003) Nutritional and physiologic significance of human milk proteins. Am J Clin Nutr 77(6):1537S-1543S

Mammary amylase: a possible alternate pathway of carbohydrate digestion in infancy. L A Heitlinger, P C Lee, W P Dillon, E Lebenthal, Pediatr Res. 171Heitlinger LA, Lee PC, Dillon WP, Lebenthal E (1983) Mammary amylase: a possible alternate pathway of carbohydrate digestion in infancy. Pediatr Res 17(1):15-18

Alpha-amylase in preterm human milk. J B Jones, N R Mehta, M Hamosh, J Pediatr Gastroenterol Nutr. 11Jones JB, Mehta NR, Hamosh M (1982) Alpha-amylase in preterm human milk. J Pediatr Gastroenterol Nutr 1(1):43-48

Proteomic characterization and comparison of mammalian milk fat globule proteomes by iTRAQ analysis. Y Yang, N Zheng, X Zhao, Y Zhang, R Han, L Ma, S Zhao, S Li, T Guo, J Wang, J Proteomics. 116Yang Y, Zheng N, Zhao X, Zhang Y, Han R, Ma L, Zhao S, Li S, Guo T, Wang J (2015) Proteomic characterization and comparison of mammalian milk fat globule proteomes by iTRAQ analysis. J Proteomics 116:34-43

Absorption and oxidation of glucose polymers of different lengths in young infants. R J Shulman, B Kerzner, H R Sloan, T W Boutton, W W Wong, B L Nichols, P D Klein, Pediatr Res. 208Shulman RJ, Kerzner B, Sloan HR, Boutton TW, Wong WW, Nichols BL, Klein PD (1986) Absorption and oxidation of glucose polymers of different lengths in young infants. Pediatr Res 20(8):740-743

Utilization of dietary cereal by young infants. R J Shulman, W W Wong, C S Irving, B L Nichols, P D Klein, J Pediatr. 1031Shulman RJ, Wong WW, Irving CS, Nichols BL, Klein PD (1983) Utilization of dietary cereal by young infants. J Pediatr 103(1):23-28

Nutrient intake and growth performance of older infants fed human milk. J E Stuff, B L Nichols, J Pediatr. 1156Stuff JE, Nichols BL (1989) Nutrient intake and growth performance of older infants fed human milk. J Pediatr 115(6):959-968

Age at weaning and infant growth: primary analysis and systematic review. B Vail, P Prentice, D B Dunger, I A Hughes, C L Acerini, K K Ong, J Pediatr. 1672e1Vail B, Prentice P, Dunger DB, Hughes IA, Acerini CL, Ong KK. Age at weaning and infant growth: primary analysis and systematic review. J Pediatr. 2015; 167(2):317-24.e1.

Contribution of villous atrophy to reduced intestinal maltase in infants with malnutrition. B L Nichols, V N Nichols, M Putman, S E Avery, J K Fraley, A Quaroni, M Shiner, E E Sterchi, F R Carrazza, J Pediatr Gastroenterol Nutr. 305Nichols BL, Nichols VN, Putman M, Avery SE, Fraley JK, Quaroni A, Shiner M, Sterchi EE, Carrazza FR (2000) Contribution of villous atrophy to reduced intestinal maltase in infants with malnutrition. J Pediatr Gastroenterol Nutr 30(5):494-502

Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the fi. eld 7 Retaining the copyright to your articleSubmit your manuscript to a journal and benefi t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the fi eld 7 Retaining the copyright to your article