Leptin is primarily secreted by adipocytes and transported into the brain to regulate food intake and energy balance 1. Several studies demonstrated significant effects of leptin on synaptic plasticity 2, neuroprotection 3, and glial cell development 4 in the central nervous system. Brain weight (wt) and total brain protein content were lower in juvenile and adult leptin-deficient mice (Lepob/Lepob, OB) than in wild-type (+/+) mice (C57BL/6, B6) 5. Decreased brain wt in OB could result from a reduction in the number of neurons and glia, decreased myelination, and a reduction in water contents 6. The adult OB brain has significantly less myelin compared with B6, with marked changes in the fatty acid composition of myelin 7. Decrease of myelination due to reduction of oligodendrocytes (OLGs) and myelin sheath (MS) is considered to be one of the causes of reduced brain wt. In the above-mentioned studies 5,6, the brains examined were from OB that were over 4 weeks old and looked obviously obese, and they reported that the brains of obese OB, which had been administered exogenous leptin from 4 to 8 weeks after birth were significantly larger than those of nontreated OB 6. The above-mentioned studies did not describe the histopathogenesis of the cerebrum during the lactation period. It is not possible to distinguish between B6 and OB pups on the basis of body wt and appearance on P7 (Table 1), although OB pups have metabolic disturbance including a thermoregulatory defect. During the lactation period, all pups are essentially hyperphagic and there is no significant difference in body wt between B6 and OB pups. As OB mice are infertile, pups of OB are usually obtained from OB-heterozygote dams, which are mated with heterozygote males under mixed condition with heterozygote and wild-type pups. In this study, however, we analyzed OB pups that had grown from eggs of OB dams that were fertilized in vitro with OB sperm. Therefore, all neonate mice were genuine OB, and thus we could analyze OB pups during the lactation period.
Myelination in the cerebra of B6, the original strain of OB, starts on postnatal day (P) 9 8 and is almost complete on P50 9. Myelin basic protein (MBP), one of MS components, was detected in the cerebra of B6 on P10 and amount of MBP reached adult level of the cerebra on P30 10, whereas the previous study 6 did not analyze leptin’s effect on myelination until 4 weeks. In contrast, leptin inhibits differentiation of glial-restricted precursor cells into OLG precursor cells (OPC) and maintains glial-restricted precursor cell in prenatal brain development 11. We reported previously that neuropeptide Y, which has a competitive effect against leptin on appetite and metabolism in adults, induced nerotrophin 3 which, by receptor-type tyrosine kinase type C phosphorylation, accelerated myelination by OLGs during the period from P7 to P14 12. Thus, we hypothesized that leptin may contribute to differentiation of OLGs and/or myelination in the early postnatal period.
We here analyzed myelination by examining morphometrically the MS in the cerebra of P28 OB and B6 using the same method as in our previous neuropeptide Y study 12. We also studied the differentiation of OLG using a histochemical technique and in-situ hybridization (ISH) of MBP mRNA in OB and B6 on P14 and P28. In this study, we replenished leptin to OB, from P7 to P28, when the cerebrum of OB is still immature. We then investigated the effects of leptin on the myelination of the cerebrum in the newborn and juvenile periods.
Materials and methods
We collected eggs from OB females and sperm from OB males (Charles River Laboratories Japan, Yokohama, Japan) and obtained fertilized OB eggs by in-vitro fertilization. We transplanted the fertilized eggs into pseudopregnant ICR mice (Jcl: ICR; CLEA Japan, Kawasaki, Japan) in which we had found vaginal plugs of males that had been operated upon for ligation of the vas deferens; the day of transplantation defined day 0 of gestation (E0). As the embryos that grew from transplanted eggs were delayed from normal development for 1 day, we obtained newborn OB by Caesarean section (CS) on E20. We prepared ICR foster mothers, which delivered newborns a few hours before the CS and put the newborn OB in the cages of foster mothers after the CS. This day was defined as P0. We thus obtained genuine OB newborns. B6 (9–15 weeks old; CLEA Japan) were mated to obtain control (+/+) neonates. The study was approved by the Ethics Committee for Animal Experimentation of Shimane University, and the animals were handled according to the institutional guidelines. We administered leptin (1 n mol/capita/day; BioVision, Milpitas, California, USA) in 0.1 ml physiological saline to OB intraperitoneally, daily at 10 a.m. from P7 to P28 in the OB+lep group. The B6 and OB were administered 0.1 ml physiological saline over the same period (the B6 and OB groups).
Four mice were anesthetized and killed in each group on P7 and P14. The total brains were removed by dissection, and the brain wt and maximum width and length of each cerebra were measured. Those for immunostaining were fixed in 100% acetone for 20 min at −20°C or 4% paraformaldehyde solution overnight at 4°C. The serial frozen sections were cut and stained immunohistochemically with anti-MBP (Chemicon International, Temecula, California, USA), anti-platelet-derived growth factor receptor-α (anti-PDGFRα; Chemicon International), anti-NG2 (Millipore, Billerica, Massachusetts, USA), anti-O4 (R and D Systems, Minneapolis, Minnesota, USA), anti-A2B5 (R and D Systems), and leptin receptor type b (obRb; Neuromics, Edina, Minnesota, USA) 12. The immunohistochemistry procedure was described elsewhere 11. The sections were observed using a confocal laser microscope (Pascal; Carl Zeiss, Jena, Germany). In a previous study in mice, myelination of the cerebral cortex began in the posterior region of the cerebrum and spread to the anterior region between P9 and P13 13. Therefore, we selected the parietal part for the present morphometric analyses. Serial coronal frozen sections of 15 μm thickness were cut, the first section was defined when the hippocampus was first observed, and then every 10 sections were collected. We analyzed a cuboid, 1.0 mm square and 1.5 mm thick, in the parietal part of the cerebrum.
Transmission electron microscopic analysis
P28 mice were anesthetized and perfused with 2.5% glutaraldehyde and 2% paraformaldehyde solution in 0.1 M PBS. After the brains were removed, 150-μm-thick coronal sections were serially cut. To analyze equivalent sections between OB and B6, we defined the section that first included the hippocampus as the first, and then we collected every second section. Five sections per brain and at least four mice per group were collected. The following procedures for transmission electron microscopic (TEM) observation were described elsewhere 12.
Analysis of MBP-mRNA expression in the cerebrum using quantitative real-time polymerase chain reaction
P14 and P28 mice were anesthetized and the whole brains were dissected. MBP-mRNA expression in the whole cerebrum was examined by QRT-PCR. Total RNA was extracted using TRI Reagent (Molecular Research Center, Cincinnati, Ohio, USA). For the reverse-transcription reaction, 200 ng of RNA was applied using Ready-To-Go RT-PCR Beads (GE Healthcare, Little Chalfont, UK). We designed the primers of MBP mRNA (+1357 to +1438; GenBank accession no. NM_001025251) containing exons 5b and 5c. The primers and the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, California, USA) were added to the reverse-transcription mixture, and the ratios of the MBP mRNA to 18S ribosomal RNA (18S rRNA; +663 to +1262; GenBank accession no. X00686) were examined by using an ABI PRISMR 7000 (Applied Biosystems). We analyzed four mice per group on P14 and P28.
Morphometric analysis of axons and nerve fiber radii by TEM
To count myelinated and nonmyelinated axons, six pictures of each ultrathin section were obtained, covering mainly the myelinated areas around the parietal part of the cingulum. We counted the axons and measured their radii and the thickness of MS at ×600. We assessed the irregularity of cross-sections by means of the circularity index [(√S1/π)/(L1/2π)] and the thickness of MS by using the G ratio (√S2/√S1) at ×4000. At least 200 myelinated fibers were analyzed per mouse, and more than three mice were examined per group. The method of morphometric analysis was essentially as reported by LoPachin et al.14.
The template of the MBP probe was 1.9 kb pairs of cDNA, and probes were labeled with digoxigenin-UTP (Roche Diagnostics, Penzberg, Germany). The ISH procedure was described in our previous paper 12.
On P7, there was no significant difference in body wt within each group. On P14, brain wt was significantly lower in OB than in B6 and OB+lep, whereas the cerebral size was not different between these groups. On P28, the brain wt and cerebral size were significantly lower in OB than in B6 and OB+lep, respectively. Table 1 summarizes the results.
We analyzed localization of obRbs in the cerebrum of B6 as wild-type mice by immunohistochemistry. ObRb were not detected in the A2B5-positive OLG progenitors 15, but obRbs were expressed on OPCs, which were NG2-positive on P7 16 (Fig. 1a and b). ObRbs were detected in the mature OLG (MO) that were O4 positive 17 and OPCs on P14 (Fig. 1c and d).
The area of MBP-positive fibers in the OB cerebrum looked smaller than that in the B6 on P28, but we could not detect any significant difference by immunohistochemical analysis between OB and OB+lep on P28 (Fig. 2a and b). In the QRT-PCR study, we checked the expression of MBP mRNA in the whole cerebrum. On P14, the index was significantly lower in OB (ratio to 18S rRNA) than in B6. It was significantly higher in OB+lep than in OB while still significantly lower in OB+lep than in B6 (Fig. 2c). On P28, the ratio was significantly higher in OB+lep than in OB and B6, whereas the ratio tended to be lower in OB but not significantly different from that in B6 (Fig. 2d). These results suggested that myelination of the cerebrum in B6 and OB was almost finished but that myelination in OB+lep still continued on P28.
TEM pictures of typical cross-sections of axons in the B6, OB and OB+lep on P28 were examined for the myelinated-axon radii (Fig. 3a–c), and the axons in the parietal part of the cerebra in these groups were counted. The axon density was significantly lower in OB than in B6, whereas it was not different between OB and OB+lep (Fig. 3d). However, the ratio of myelinated axons to total axons was significantly lower in OB than in B6, and higher in OB+lep than in OB (Fig. 3e). The average radius of the myelinated axons in the parietal part of the cerebrum was significantly larger in OB than in B6 or in OB+lep; the difference between B6 and OB+lep was not significant (Fig. 3f). The circularity indexes of nerve fibers in the B6 (77.2±1.5%), OB (76.1±1.0%), and OB+lep (76.6±1.3%) were each over 75%, which allowed us to assume these nerve fibers as circles. We thus could evaluate leptin’s effect on myelination using the G ratio (axon radius/fiber radius). The average G ratio was significantly larger in B6 than that in OB or OB+lep, but it was not significantly different between OB and OB+lep (Fig. 3g).
To examine the OLG differentiation, we analyzed the numbers of OPCs and MOs using immunohistochemistry and ISH. On P14, PDGFRα-positive OPCs before the MO stage were located in the parietal part of the cerebra of B6 and OB 18 (Fig. 4a and b). On P28, MBP mRNA-expressing OLGs that were MOs were detected in the same part of the cerebra in B6 and OB (Fig. 4c and d). On P14, there were significantly fewer OPCs in the cerebra in OB and OB+lep than in B6 (Fig. 4e). There was no significant difference in the number of OPCs between OB and OB+lep on P14. The number of MOs in the cerebrum was significantly higher in OB and OB+lep than in B6 on P14. There was no significant difference in the number of MOs between OB and OB+lep on P14. On P28, there were significantly fewer MOs in the cerebrum in OB and OB+lep than in B6 (Fig. 4f). On P28, OB had the same number of MOs as OB+lep. B6, OB, and OB+lep each had a significantly greater number of MOs on P28 than on P14.
We reported that the replenishment of leptin in OB during the embryonic period using an ex-utero method was effective for glial differentiation 19, but we did not detect leptin’s effects on glial differentiation after birth in the present study. In our previous in-vitro analysis of neurosphere, leptin maintains its self-renewal ability and the reactivity of epidermal growth factor in immature neural lineage cells, and the signal is mediated, at least in part, by the PI3 K pathway 20.
Leptin administration to OB, beginning at the age of 4 weeks and lasting for 6 weeks, recovered brain wt and protein to the same levels as in B6 6. Leptin administration induced increases in GAP-43 and Syntaxin1, which are components of axons and synapses, and was suggested to induce the elongation of axons and the increase in synapses 21. Leptin administration to OB after P28 increased total brain DNA content and cell numbers 22. In the above-mentioned studies, leptin injection to OB started after P28 when OB obviously looked obese. As MBP reached an adult concentration in B6 on P30 9, leptin replenishment in mice after 4 weeks old was unable to clarify leptin’s role in myelination.
We examined the effect of leptin on the brain development in lactation period. Brain wts were significantly lower in OB than in B6 on both P14 and P28. Leptin administration to OB from P7 to P28 recovered the brain wt of OB. The density of nerve fibers was significantly lower in OB than in B6 on P28, and leptin injection did not affect the density of OB. The ratio of myelinated axons to total axons was significantly lower in OB than in B6 on P28, and the ratio in OB+lep was restored to the B6 level on P28. The average radii of myelinated axons of OB+lep was also restored to the B6 level on P28. The G ratio, which is related to the thickness of MS, was significantly lower in OB than in B6 on P28. However, leptin injection did not affect the G ratio in OB+lep. These results suggested that the increase in MBP mRNA by leptin injection to OB increased the density of myelinated axons but did not increase the thickness of MS.
It was reported that leptin administration significantly increased the preservation of caudal white matter after 28 days of spinal cord injury because of continuation of myelin synthesis and because mRNA of peroxisome proliferator activated receptor (PPAR) α, which is a nuclear transcription factor and has anti-inflammatory effect, significantly increased in caudal injured spinal cord after leptin injection 23. PPARα and PPARγ were present in C6 glioma cells, which have a potential to differentiate into OLGs 24. PPARγ affected differentiation of C6 glioma cells and PPARα-induced myelin synthesis after differentiation in-vitro study 24. In the present study, obRbs were detected on the OPCs and MOs in the cerebra, excluding undifferentiated glial cells. In addition leptin replenishment affected increase of myelination but differentiation of OLG. Taken together, effect of leptin on myelin synthesis in the present study may be mediated through induction of PPARα.
The present findings suggest that leptin has an effect on myelination, particularly on the density of myelinated axons in the cerebrum, but does not affect OLG differentiation after birth.
This work was supported by JSPS KAKENHI Grant Number 15209034. The authors thank Y. Takeda (Department of Developmental Biology, Faculty of Medicine, Shimane University) for the histological preparations, and Y. Okui and T. Yoneyama (Center for Integrated Research in Science, Shimane University) for the TEM preparations.
Conflicts of interest
There are no conflicts of interest.
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