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Effect of Lactobacillus rhamnosus GG supernatant on serotonin transporter expression in rats with post-infectious irritable bowel syndrome

AIM To evaluate the effect of Lactobacillus rhamnosus GG supernatant (LGG-s) on the expression of serotonin transporter (SERT) in rats with post-infectious irritable bowel syndrome (PI-IBS). METHODS Campylobacter jejuni 81-176 (1010 CFU/mL) was used to induce intestinal infection to develop a PI-IBS model. After evaluation of the post-infectious phase by biochemical tests, DNA agarose gel electrophoresis, abdominal withdrawal reflex (AWR) test, and the intestinal motility test, four PI-IBS groups received different concentrations of LGG-s for 4 wk. The treatments were maintained for 1.0, 2.0, 3.0 or 4.0 wk during the experiment, and the colons and brains were removed for later use each week. SERT mRNA and protein levels were detected by real-time PCR and Western blot, respectively. RESULTS The levels of SERT mRNA and protein in intestinal tissue were higher in rats treated with LGG-s than in control rats and PI-IBS rats gavaged with PBS during the whole study. Undiluted LGG-s up-regulated SERT mRNA level by 2.67 times compared with the control group by week 2, and SERT mRNA expression kept increasing later. Double-diluted LGG-s was similar to undiluted-LGG-s, resulting in high levels of SERT mRNA. Triple-diluted LGG-s up-regulated SERT mRNA expression level by 6.9-times compared with the control group, but SERT mRNA expression decreased rapidly at the end of the second week. At the first week, SERT protein levels were basically comparable in rats treated with undiluted LGG-s, double-diluted LGG-s, and triple-diluted LGG-s, which were higher than those in the control group and PBS-treated PI-IBS group. SERT protein levels in the intestine were also comparable in rats treated with undiluted LGG-s, double-diluted LGG-s, and triple-diluted LGG-s by the second and third weeks. SERT mRNA and protein levels in the brain had no statistical difference in the groups during the experiment. CONCLUSION LGG-s can up-regulate SERT mRNA and protein levels in intestinal tissue but has no influence in brain tissue in rats with PI-IBS.

INTRODUCTION
Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder (FGD), annually affecting 12% to 30% of the population worldwide [1][2][3][4][5] . IBS can be divided into four subtypes, namely, IBS with constipation (IBS-C), IBS with diarrhea (IBS-D), mixedtype IBS (IBS-M), and untyped IBS (IBS-U) [6][7][8] . Acute infectious gastroenteritis (IGE) is an important risk factor for developing IBS, with 5% to 31% of the patients developing post-infectious IBS (PI-IBS) [9][10][11][12] . Recent reports indicate that abnormalities in serotonergic signaling systems are involved in the development of PI-IBS, particularly those affecting serotonin (5-HT) levels in the gastrointestinal tract [13][14][15] . As a signal transducer and neurotransmitter, 5-HT modulates intestinal fluid secretion, gut motility, and gastrointestinal sensation [16] . Serotonin transporter (SERT) is a universally existing transmembrane transport protein that plays a key role in 5-HT reuptake [15,17] . SERT has two important polymorphic areas. The first is named 5-HT-transportergene-linked polymorphic region (5-HTTLPR), which is located in the regulatory region of its gene (SLC6A4; chromosome 17q11.1-q12) [18] . The most frequently studied variant is subdivided into long (L) and short (S) alleles [19,20] . The transcriptional efficiency of the L/L genotype is significantly higher than that of the L/S and S/S genotypes [21] . Furthermore, the frequency of the L/L genotype in C-IBS was significantly higher than that in D-IBS, and the S/S genotype is higher in D-IBS [21][22][23][24] . In other words, the expression of SERT is higher in C-IBS, and lower in D-IBS. The second is named STin2, which is also called variable number tandem repeats (VNTR). Wang et al [25] have found a higher ratio of STin2.12/10 and a lower ratio of STin2.12/12 in IBS patients, and there were no significant differences between different subtypes. However, a small-scale study on SERT in PI-IBS, conducted by Wheatcroft et al [26] , showed that SERT expression was reduced in PI-IBS patients.
Lactobacillus rhamnosus GG (LGG) is the best studied member of the lactic acid bacteria and is known to have positive effects on human health [27] . Probiotics such as LGG are potential treatment options in patients with IBS [28] .
LGG could exclude pathogens, promote mucosal immunity against Salmonella infection [29] , and reduce the rotavirus-related diarrhea by increasing the levels of interferon-γ (IFN-γ) [30] . The ESPGHAN Working Group recommends using LGG for preventing nosocomial diarrhea [31] . Furthermore, LGG reduces the frequency and severity of abdominal pain in children with IBS [32] , along with improving disease severity, especially in IBS-D and IBS-A subtypes [33] .
Our previous study had confirmed that Lactobacillus rhamnosus GG supernatant (LGG-s) could up-regulate SERT mRNA and protein levels in intestinal epithelial cells and mouse intestinal tissues [34] . The aim of this study was to investigate the effects of LGG-s on the expression of SERT mRNA and SERT protein (SERT-P) in the colon and brain in a rat model of PI-IBS.

Bacterial culture, LGG-s, and Campylobacter jejuni
LGG [53103, American Type Culture Collection (ATCC), United States] was incubated in Lactobacillus MRS broth (Oxoid CM0359) at 37 ℃ for 24 h, according to ATCC guidelines, diluted in MRS broth, and cultured again at 37 ℃ to reach log phase with the density determined as 0.5 at A600 [34] . The culture suspension was centrifuged at 4000 g for 15 min, then the supernatant was collected and filtered through 0.20-μm filters [35] .  [36] .

Animal studies
The study was performed on male Sprague-Dawley rats (aged between 7 and 8 wk) obtained from Laboratory Animal Center of Chinese People's Liberation Army General Hospital (Beijing, China). The rats were kept under the following conditions: the temperature of 23 ℃ ± 1 ℃, a 12h/12 h light/dark cycle (lighting from 08:00 to 20:00), and free access to a sterile diet. The rats were then divided into two groups. The first group of rats were designated as the control group (n = 25, normal and healthy), which was given PBS (2 mL/d per rat), and another group as the model group of PI-IBS (n = 85), which was given C. jejuni (10 10 CFU/mL, 2 mL/d per rat) through an intra-gastric needle (Thermo Fisher Scientific, Hampton, United States). The gavage continued for 7 d. Then, the stool culture, body weight of the rats, and relative content of stool water were tested to evaluate the phase of infection. If the rats get rid of infection with a higher Bristol score of faeces, faster intestinal transit, and visceral hypersensitivity, they were considered to enter the post-infectious phase as PI-IBS. After the model evaluation, the rats were regrouped; the control group was designated as M (n = 20) and given PBS, and the PI-IBS model group was divided into four groups and given PBS, undiluted LGG-s, double diluted LGG-s, and triple diluted LGG-s, respectively (A, B, C and D, respectively, n = 20) through a gavage needle [34] . PBS or LGG-s was administered at 2 mL/d/ per rat, and the treatments were maintained for 1.0, 2.0, 3.0 or 4.0 wk during the experiment. Five rats of each group were sacrificed; the colons and brains were removed for later use each week.
During all experiments, housing and diet conditions were the same for all groups. Rats infected with C. jejuni were housed in another room to prevent crosscontamination from infected to uninfected. The protocol was approved by the Animal Use and Care Committee of Tianjin Medical University ( Figure 1).

Campylobacter gavage
Before the infection, all rats received 1 mL of 5% (w/v) bicarbonate solution via a ball-tipped inoculating needle to neutralize the gastric acid. Thirty minutes later, C. jejuni in 2 mL of PBS was given to the PI-IBS model group and 2 mL of sterile PBS to the control group [37] .

Assessment of acute colonization by C. jejuni
The fresh stool specimens were cultured for the presence of C. jejuni on Campylobacter selective agar plates using biochemical tests and DNA agarose gel electrophoresis, and the general condition, body weight, and relative content of stool water were observed or tested on the LTD, China) and tested by agarose gel electrophoresis.
For measuring the relative content of stool water, fresh stools were obtained from rats under manual restraint by spontaneous or perianal-stimulated defecation, weighed, and then dried at 50 ℃ for 72 h followed by room temperature for 48 h. Dry stools were weighed again to determine the percent wet weight of stool.

Determination of PI-IBS
In the case of two consecutive tests with negative culture, rats were considered to get rid of infection. Rats without infection were kept separately from infected rats. After all of the rats no longer had detectable C.
bowel along which it contains the activated carbon and the length of the whole bowel were measured, and the ratio of these two lengths was taken as the intestinal transit rate (ITR) [40] .

Real-time polymerase chain reaction
To evaluate the levels of SERT mRNA after treatment with LGG-s, colon and brain samples were harvested. Rats were sacrificed by cervical dislocation, and total RNA was prepared from 50 mg of tissue from each rat with Trizol, according to the manufacturer's instructions (Life, Hilden, Germany). An iScriptcDNA synthesis kit (Bio-Rad Laboratories, Inc., Hercules, CA, United States) was used to synthesize the cDNA. The PCR were set up in a volume of 20 μL containing 1.0 μL cDNA, 10 μL 2 × iQSYBR Green Supermix (Roche Applied Science), and 0.6 μL both forward and reverse primers, replenished with DEPC treated ddH2O. Quantitative RT-PCR was performed on an ABI One plus setup PCR thermocycler. The sequences of primers are given in Table 1. Glyceraldehyde-3phosphate dehydrogenase (GAPDH) was measured as an internal control. Relative mRNA expression was calculated using the 2 -ΔΔCt method.

Western blot analysis
Proteins were extracted from colonic and cerebral tissues, and protein levels were quantified using a BCA kit (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China), according to the manufacturer' s instructions. The protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% nonfat milk, incubated with a primary antibody (SERT antibody: dilution 1:5000, EPR12735, Abcam Biotech jejuni in the stool, they were considered to be in the post-infectious time period. Fresh stool was collected for 3 consecutive days from all rats and graded by a modified Bristol Stool score [38] . Normal stool was graded as 1, soft and poorly formed stool graded as 2, and watery stool as 3. Five rats from the control group and four from the model group were randomly chosen to perform the tests. Visceral hypersensitivity was evaluated by abdominal withdrawal reflex (AWR) test, and the intestinal motility was detected.
AWR experiment was done as previously reported [39] . Rats were fasted for 18 h before the test was performed. The night before AWR, balloons (7-8 mm diameter) were inflated overnight to stretch the latex, then the balloons became compliant. Following anesthesia by ether inhalation, a balloon coated with paraffin oil was inserted into the rectum with the tail of the balloon, 1 cm from the anus, fixed at the base of the tail. The balloon was connected via a double barreled cannula, with one joint connected to the air pump and another connected to the pressure gauge. Rats were given 30 min to accommodate the environment. Then, the balloon was distended at the pressures of 20, 40, 60 and 80 mmHg. Distention was sustained for 15 s, at intervals of 10 min. The distention was performed three times at each pressure, and the AWR scores were recorded. AWR was done by researchers who had no understanding of the experiment.
Intestinal motility was detected by activated carbon solution gavage. Before the experiment, rats were fasted for 24 h and then given 2 mL of 10% activated carbon solution through an inoculating needle. After 50 min, rats were sacrificed by cervical dislocation. Then, a laparotomy was performed, and the whole bowel was taken out and moistened with PBS. The bowel was freely flattened on the table. The length of the

Statistical analysis
Statistical analyses were carried out using SPSS 22.0 (SPSS, Chicago, IL, United States). Quantitative data are expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) and post hoc tests (LSD and Dunnett's T3) were used to compare the values of quantitative RT-PCR. For all analyses, P < 0.05 was defined as statistical significance.

Campylobacter colonization phase
A total of 110 rats were used in the study, none showed C. jejuni infection prior to the first gavage (as determined by stool culture), and all rats inoculated with C. jejuni exhibited C. jejuni colonization within 3 d after gavage (1 rat died from severe gavage trauma). Thus, a total of 109 rats (25 control rats and 84 PI-IBS model rats) were included before the model evaluation.
Three to fourteen days after infection, rats in the control group had good spirits, normal activity, and glossy hair. However, rats in the PI-IBS model group were decadent, indolent, and lackluster. The general conditions were returned to normal in about 42 d.
The rat weight in the model group was significantly lower than that in the control group at the 3 rd , 7 th , 14 th and 28 th days (P < 0.05). After that, the weight in the model group was still lower than that in the control group with no statistical significance (P > 0.05).
The relative content of water in the fresh stool in the model group was significantly higher than that of the control group at the 3 rd , 7 th , 14 th , 28 th and 70 th days (P < 0.05). At the 42 th and 56 th days, the model group was a little higher than the control group ( Figure 2).

Post-infectious phase
As two consecutive tests resulted in negative culture, the PI-IBS. The 3-d average Bristol score of stool and the intestinal transit rate were higher in the model group than in the control group (P < 0.05). AWR score of rats in the model group was also higher than that of the control group. However, there was no statistical difference between the two groups at the pressures of 20 and 80 mmHg (P = 0.12 and 0.06, respectively) ( Figure 3).

Effects of LGG-s on SERT mRNA and SERT-P expression in rat intestinal tissues
For the PI-IBS PBS gavage group (A), the levels of SERT mRNA expression were lower than those of the control group (M) throughout the experiment. However, only at the 3 rd week, SERT mRNA expression in group A was significantly lower than that in group M by 0.7-fold (P = 0.023, P < 0.05). Undiluted LGG-s (B) up-regulated SERT mRNA expression level by 1.8-fold compared with group A by the 1 st week (P = 0.002), and the level was slightly higher than that of group M (P > 0.05). By the end of the 2 nd week, the level of SERT mRNA in group B was 2.67 times and 3.7 times higher than those in groups M and A, respectively (P = 0.018 and 0.012, respectively). SERT mRNA expression in group B increased continuously, and at the 3 rd week, it was 4.4-fold, 6.1-fold, and 1.7-fold more than those in groups M, A, and D (triple diluted LGG-s), respectively (P = 0.003, 0.003 and 0.028, respectively), although there was no significant difference between groups B and C (double diluted LGG-s). By the last week, the level of SERT mRNA in group B was only 2.1-fold higher than that in group A (P = 0.016). Double diluted LGG-s (C) moderately increased SERT mRNA levels by 1.8-fold compared with group A by the 1 st week (P = 0.027). The level of SERT mRNA in group C was 2.3-fold higher than that of group A at the 2 nd week (P = 0.012), and 4.6-and 6.4-fold at the 3 rd week, and 1.9-and 2.6-fold at the last week compared to groups M and A (P = 0.012, 0.009, 0.032, and 0.011, respectively). However, there was no statistical significance between groups C and D at the 2 nd week (P > 0.05).
Triple diluted LGG-s (D) significantly up-regulated SERT mRNA expression levels by 6.9-, 9.4-, 5.3-and 5.1-fold compared to groups M, A, B, and C at the end of the first week (P = 0.02, 0.01, 0.002, and 0.002, respectively), but the level decreased rapidly at the end of the 2 nd week. The SERT expression levels were similar between the 2 nd week and the 3 rd week, which were 2.3-and 2.5-fold higher compared to group M (P = 0.015 and 0.001, respectively) and 3.1-and 3.5-fold higher compared to group A (P = 0.009 and 0.028, respectively).  The variation tendency of SERT-P level was similar to that of SERT mRNA. SERT-P level was lower in group A than in group M during the whole experiment, and there was no significant difference only at the 3 rd week (P = 0.177). SERT-P level was similar between groups B and A, although it was lower in group B than in all other groups, at the 1 st week. SERT-P level was comparable in groups B, C, and D by the 2 nd and 3 rd weeks. SERT-P level was slightly higher in groups B, C, and D than in group M at the 4 th week, but there was no statistical significance ( Figure 4).

Effect of LGG-s on SERT mRNA and SERT-P expression in rat brain tissues
There were no statistical differences in SERT mRNA or SERT-P expression between the different dilution concentrations. The levels of SERT mRNA in groups B, C, and D had a little increase compared to groups M and A at the 1 st week, but turned lower at the last week. Double diluted LGG-s resulted in a minuscule increase in SERT-P at the 3 rd week, which declined at the end of the study ( Figure 5).

DISCUSSION
According to the Rome Ⅳ criteria, IBS is one of the lower gastrointestinal tract disorders, which identify about two thirds of suffers, along with requiring that the pain either be relieved by defecation or associated with changes in stool frequency or/and consistency [8,41,42] . PI-IBS often exhibits characteristics of diarrhea, and patients often have a history of acute gastrointestinal infection [9][10][11][12] .
Many studies showed that infection with C. jejuni strain correlates with the development of PI-IBS [43][44][45] . C. jejuni produces a range of toxins including cytolethal distending toxin, which first produces a secretory diarrhea in the small intestine, subsequently invading the distal ileum and colon to produce an inflammatory ileocolitis [46] . Therefore, C. jejuni was used to build a model of PI-IBS in this study. The mechanisms that underlie chronic disturbance of gut function are thought to involve chronic microscopic mucosal inflammation, visceral hypersensitivity, dysregulation of gut microbiota, and abnormal neuromuscular function [47][48][49][50] . 5-HT was found in the gastrointestinal tract and central nervous system (CNS). It functions both as a neurotransmitter and as a local hormone in the peripheral vascular system in the gut [51] . About 95% of body 5-HT is found in the gastrointestinal tract, with 90% in enterochromaffin cells (EC cell) and 10% in serotonergic neurons of myenteric plexus [51,52] . As a signal transducer and neurotransmitter, 5-HT is a key molecule, regulating visceral perception and intestinal motility [53] . 5-HT exerts its action by binding to its receptors (5-HT1 to 5-HT7) present in both intrinsic and extrinsic primary afferent neurons [54] . Serotonin receptors that are known to affect gut motor functions are those belonging to the 5-HT1, 2, 3, 4 and 7 subtypes [55][56][57] . 5-HT receptors contract effector cells when bound by 5-HT2A and relax cells by 5-HT4 and 5-HT7 subtypes [58] . Neuronal 5-HT3 leads to increased release of acetylcholine from cholinergic neurons [52] . The release of 5-HT acting on effector cells leads to secretory reflexes, peristaltic reflexes, and if superfluous, diarrhea, abdominal pain, or visceral hypersensitivity [59] . Clinical trials have shown an increased level of 5-HT in IBS [60][61][62] . Serotonin reuptake transporter (SERT) plays an irreplaceable role in 5-HT inactivation by decreasing the content of 5-HT in the synaptic cleft [63] . SERT on the cell membrane of enterocytes is vital to transport 5-HT into the cell, with 5-HT metabolized by monoamine oxidase [64] . SERT was expressed by nearly all of the intestinal epithelial cells on the surface of the lumen [65] . Biochemical abnormalities in PI-IBS patients including hyperplasia of the EC cells and depressed 5-hydroxyindole acetic acid/5-HT ratio suggested impaired SERT function [44] . Many researchers have demonstrated that IBS patients have a remarkably lower level of SERT expression in the intestine [66] . Coates et al [17] first demonstrated a significantly decreased level of SERT in IBS. SERT expression can be regulated by a series of factors, such as gene polymorphisms, microRNAs, immunity, inflammation, gut microbiota, and growth factors [67] . The SERT gene, SLC6A4, containing 5-HTTLPR [19] , VNTR STin2 [68] , and functional single nucleotide polymorphisms (SNPs), has a positive association with etiology of IBS [69] . 5-HTTLPR is the most frequently studied, which is subdivided into long (L) and short (S) alleles [19] .
Previous studies found that the frequency of L/L genotype is significantly higher in C-IBS than those of L/S and S/S genotypes [21] . VNTR showed a higher ratio of STin2.12/10 and a lower ratio of STin2.12/12 in IBS [25] . Immune activation of the gut mucosa plays a critical role in EC cell hyperplasia and reduced SERT activity in PI-IBS [70,71] . Foley et al [72] found that SERT mRNA had a lower level in D-IBS, which was correlated with increased numbers of mucosal intraepithelial lymphocytes and mast cells. Pro-inflammatory mediators, such as IFN-γ and tumor necrosis factor-α, reduce the expression of SERT mRNA, SERT-P, and SERT function in Caco-2 cells [72] . However, a protective cytokine, transforming growth factor-β1, could rapidly activate SERT activity and inhibit intestinal inflammation via PI3K and syntaxin 3 [73] . The acute infection of C. jejuni produces diarrhea in the small intestine, and leads to the inflammatory response, which increases the ratio of pro-/anti-inflammatory factors. In other words, the SERT mRNA and SERT-P levels in intestinal tissues were significantly decreased in the PI-IBS PBS group than in controls. The highest concentration of LGG-s did not induce the highest expression of SERT, which is inconsistent with a previous study which found a dosedependent up-regulation of expression of SERT [34] . This may due to the differences in the contents of various substances (proteins, fatty acids, inorganic salts, etc.) in the supernatant, which, combined with previous C. jejuni infection, could lead to different immune activation.
In addition, gut host-microbial interactions are important factors in IBS. Studies have found that Lactobacillus, Bifidobacterium, Actinobacteria, and Bacteroidetes were decreased [74][75][76] , while Proteo bacteria, Firmicutes, and Firmicutes/Bacteroidetes ratios were increased in fecal samples of IBS-D patients [77] . Enteropathogenic E. coli and E. coli Nissle 1917 could decrease SERT mRNA and increase 5-HT bioavailability [78,79] . Our previous study proved that LGG-s could up-regulate the SERT mRNA and SERT-P levels in enterocytes and mouse intestinal tissues [34] .
In this study, we also proved that LGG-s had a positive effect on SERT expression in colon tissues in PI-IBS rats. It may provide a novel solution for the treatment of IBS. Further studies are needed to find whether LGG-s has any impact on the composition of rat gut microbiota.
Growth factors, such as epidermal growth factor, basic fibroblast growth factor, and nerve growth factor, may be involved in the up-regulation of SERT expression [80][81][82] . A protein, known as p40, expressed by LGG, activates epidermal growth factor receptor (EGFR) [35] , which might activate the mechanism of LGG-s induced up-regulation of SERT expression.
Gender may be another factor. Although IBS is an universally disease, women are more likely to suffer from this illness than men [83] . It has been found that SERT mRNA levels in the rectal mucosa of women with IBS-D were higher than those in men. Female SERT knockout rats showed remarkable visceral hypersensitivity than male rats [84] . However, male animals were also commonly used in PI-IBS research [85,86] . Ibeakanma et al [87] found that brain-gut interactions could exaggerate peripheral nociceptive signaling in male mice with PI-IBS. An experimental model of male mouse induced by stress or Giardia also showed prominent visceral hypersensitivity [88] . As for this study, male rats were selected to perform the research.
Alterations in the bidirectional interactions between the gut and the nervous system play an important role in IBS pathophysiology and symptom generation. Communication between the gut and the CNA, both in the ascending (gut-to-brain) and descending (brainto-gut) directions, is called the gut-brain axis [89] . Gut microbes may communicate with the gut-brain axis via production of neuro-active and neuroendocrine molecules such as serotonin, aminobutyric acid (GABA), histamine, noradrenaline, and adrenaline [90] . Lactobacilli can convert glutamate into GABA [91] , and administration of L. rhamnosus JB-1 to mice altered the patterns of GABA receptors in the brain [92] . There is very little study about the changes of SERT in the CNS. However, no significant differences were found in SERT expression between the LGG-s treated group and the control group in this study. It is possible that the signal might be prevented from entering the brain by the bloodbrain barrier (BBB) [93] . The connections between cells in the BBB are tighter, which greatly limits the endothelial permeability via para-cellular and trans-cellular transport pathways [94] . The exchange of substances in the blood and brain is mainly accomplished by various transporters expressed in vascular endothelial cells [95] .
Regulators or neurotransmitters, secreted by LGG, may lack specific transporters. Because of the BBB, it is very difficult for 5-HT in the blood to enter the CNS, whereas 5-HT in the central and peripheral nerves is a separate system with different functions. Therefore, as a reuptake transporter, SERT may also be regulated in different ways. The effect of LGG injected directly into the brain is still need to be studied.
In conclusion, we found that LGG-s up-regulated SERT expression in intestinal tissues, but had no statistical effect in brain tissues in PI-IBS rats. By decreasing 5-HT levels, LGG-s may be a potential strategy for helping improve clinical symptoms of IBS.

Research background
Probiotics have been approved to be used to relieve irritable bowel syndrome (IBS), and Lactobacillus rhamnosus GG (LGG) is the best studied member of lactic acid bacteria and has supportive therapeutic efficacy in IBS. However, the mechanism remains a significant challenge to researchers. This study developed a PI-IBS model to evaluate the effect of LGG supernatant on serotonin transporter expression.

Research motivation
This study is a part of a National Natural Science Foundation of China project. On the basis of developing an experimental model of PI-IBS, this research explored the effect of LGG-s on SERT levels in intestinal and brain tissues.

Research objectives
This study detected the expression levels of SERT mRNA and SERT-P to evaluate the effect of LGG-s in PI-IBS rats, which were infected with C. jejuni.
LGG-s could up-regulate SERT mRNA and SERT-P levels in rat intestinal tissues but had no influence in rat brain tissues. The more detailed research on LGG-s will contribute to more accurate treatment of IBS.

Research methods
The model group of PI-IBS (n = 85) was given C. jejuni (10 10 CFU/mL, 2 mL/d per rat) for 7 d, then the body weight of the rats and the relative content of stool water were measured to evaluate the phase of infection, and the fresh stool specimens were cultured for the presence of C. jejuni on Campylobacter selective agar plates. After the model evaluation, the rats were regrouped, and each group was gavaged with different concentrations of LGG-s. The treatments were maintained for 1.0, 2.0, 3.0 or 4.0 wk during the experiment. Then, SERT expression was detected by RT-PCR and Western blot to evaluate the effect of LGG-s.

Research results
The levels of SERT mRNA and SERT-P in intestinal tissues were up-regulated by treatment with LGG-s of different concentrations. Triple-diluted LGG-s showed a more significant difference within a short term, while, in the long run, undiluted and double-diluted LGG-s proved better. However, there were no significant differences in SERT mRNA and SERT-P in the brain tissues between each group, with or without treatment with LGG-s. Some factors and differences in the contents of various substances (proteins, fatty acids, inorganic salts, etc.) in the supernatant may induce a different increase in SERT levels. More detailed research about LGG-s is needed.

Research conclusions
This study demonstrates that LGG-s up-regulates SERT expression in intestinal tissues, but has no statistical effect in brain tissues in PI-IBS rats. The previous study has proved that LGG-s could up-regulate the SERT levels in intestinal tissues in healthy mice. Moreover, LGG-s led to dose-dependent expression of SERT. The contents of substances in the supernatant, combined with their different concentrations, molecular mass, and previous C. jejuni infection, may result in this phenomenon. Therefore, more detailed research about LGG-s and relief of clinical symptoms with the treatment of LGG-s would be done in our next work.

Research perspectives
The infection with C. jejuni could help to build a PI-IBS model with lower expression of SERT. For the future accurate treatment of IBS, proteomics analysis of LGG-s is important and urgent.


Axonal abnormalities in vanishing white matter

Abstract Objective We aimed to study the occurrence and development of axonal pathology and the influence of astrocytes in vanishing white matter. Methods Axons and myelin were analyzed using electron microscopy and immunohistochemistry on Eif2b4 and Eif2b5 single‐ and double‐mutant mice and patient brain tissue. In addition, astrocyte‐forebrain co‐culture studies were performed. Results In the corpus callosum of Eif2b5‐mutant mice, myelin sheath thickness, axonal diameter, and G‐ratio developed normally up to 4 months. At 7 months, however, axons had become thinner, while in control mice axonal diameters had increased further. Myelin sheath thickness remained close to normal, resulting in an abnormally low G‐ratio in Eif2b5‐mutant mice. In more severely affected Eif2b4‐Eif2b5 double‐mutants, similar abnormalities were already present at 4 months, while in milder affected Eif2b4 mutants, few abnormalities were observed at 7 months. Additionally, from 2 months onward an increased percentage of thin, unmyelinated axons and increased axonal density were present in Eif2b5‐mutant mice. Co‐cultures showed that Eif2b5 mutant astrocytes induced increased axonal density, also in control forebrain tissue, and that control astrocytes induced normal axonal density, also in mutant forebrain tissue. In vanishing white matter patient brains, axons and myelin sheaths were thinner than normal in moderately and severely affected white matter. In mutant mice and patients, signs of axonal transport defects and cytoskeletal abnormalities were minimal. Interpretation In vanishing white matter, axons are initially normal and atrophy later. Astrocytes are central in this process. If therapy becomes available, axonal pathology may be prevented with early intervention.

Introduction
Vanishing white matter (VWM) commonly presents in early childhood, but onset may vary between antenatal period to senescence. 1 Age of onset is inversely related to clinical severity. 2 Clinically, VWM is characterized by chronic neurological decline with additional episodes of rapid, severe deterioration provoked by stressors like febrile infections and minor head trauma, which may end in coma and death. [3][4][5][6] As yet, no curative treatment is available.
VWM is caused by mutations in the genes encoding the five subunits of the eukaryotic translation initiation factor 2B (eIF2B), EIF2B1 through EIF2B5. 7,8 eIF2B is an enzyme complex essential for translation of mRNAs into proteins and central in downregulating the rate of translation under different stress conditions. 9 It is unclear why mutations in this housekeeping factor lead to a disorder specifically affecting the brain white matter, whereas other organs are mostly spared. 10 The neuropathological findings in VWM include diffuse lack of myelin and cystic degeneration of the cerebral white matter. 5,11 U-fibers, corpus callosum, internal capsule, and brainstem are better preserved. 5,6,12 Considering the degree of cerebral white matter damage, reactive astrogliosis is disproportionately meager. 13,14 Astrocytes are dysmorphic and immature. [13][14][15] In relatively preserved white matter areas, lack of myelin is associated with increased numbers of oligodendrocyte progenitor cells (OPCs), while mature, myelinating oligodendrocytes are scarce. 13,16,17 Axons are lost in cavitated white matter and their numbers are variable in less severely involved areas. 5,6,12,13,18 Remaining axons have been reported as abnormally thin. 6 Axonal swellings and spheroids have been described around cavitated areas and in the subcortical white matter. 19,20 Otherwise, axons have been reported as microscopically intact with a normal cytoskeleton at ultrastructural level. 13,19,21 Cortex and central gray matter structures remain largely preserved without evident neuronal loss. 5,6,13,14,18,20 White matter astrocytes and oligodendrocytes are selectively affected. 11,14,15,22 Recent studies indicate that astrocyte dysfunction is primary and oligodendrocyte dysfunction secondary. 16,23 As astrocytes and oligodendrocytes are important for axonal support, their dysfunction in VWM may affect axonal integrity and function, but a primary axonal defect in VWM has not been ruled out. [24][25][26] In this study, we investigated axonal morphology in brain tissue of VWM patients and mutant mice, in the latter at different time points, and used primary cell cultures to assess whether axonal abnormalities are primary or secondary to abnormality of VWM astrocytes.

Animals
Mouse models (background strain C57Bl/6J) were generated by inserting a point mutation in Eif2b4 (Eif2-b4Arg484Trp/Arg484Trp) or Eif2b5 (Eif2b5Arg191His/ Arg191His) and bred into homozygous single mutants as well as heterozygous-homozygous double-mutants, which in increasing order of severity are referred to as, respectively, 2b4 ho , 2b5 ho , and 2b4/2b5 heho mice. 23 These mice replicate the human disease in clinical and pathological features and in variation in severity. Timed mating was performed with wild-type or mutant mice, with the day of plugging denoted as embryonic day 0 (E0).

Cell culture
Astrocytes or forebrain mixed cells were isolated from cerebral hemispheres of E18 mice and cultured following previously described methods with minor modifications. [27][28][29][30][31] Cultures were kept in a humidified incubator at 37°C and 7.5% CO 2 at all times. For astrocyte cultures, cells were passaged two times, frozen and stored in liquid nitrogen until further use. Before starting a co-culture, astrocytes were first plated into a flask and cultured for 1 week in order to let them recover. Astrocyte-forebrain co-cultures were performed in poly-L-lysine-coated 96well plates (Greiner). Fifteen thousand astrocytes were plated per well and cultured until confluence (4 days), after which 15.000 forebrain mixed cells, including neurons, oligodendrocytes, microglia, and a few astrocytes per well were plated on top of the astrocytes. Multiple wells with astrocytes were maintained without forebrain cells in order to determine astrocyte monolayer purity. As previously described, for the first 11 days, the co-culture medium contained insulin, followed by 17 days without insulin but with triodothyronine (T3, Sigma-Aldrich). Half of the medium was replaced with fresh medium 3 times a week. Experiments were performed at least four independent times with at least three replicates per combination of astrocyte-forebrain co-culture.

Immunocytochemistry
Cells were fixed with 4% paraformaldehyde (PFA, Thermo Scientific) and 4% sucrose (Sigma-Aldrich) for 20 min at room temperature, washed two times with PBS, and permeabilized and blocked with 0.5% Triton X100 (Sigma-Aldrich) and 10% normal goat serum (Gibco) in PBS for 1 h at room temperature. Subsequently, cells were stained overnight at 4°C with primary antibodies in staining solution containing PBS, 0.1% Triton X100, and 10% normal goat serum. Then, cells were washed three times with PBS, followed by a 2-h incubation of secondary antibodies in staining solution. Finally, cells were washed two times with PBS and stored in PBS at 4°C until further analysis.
Primary antibodies used were directed against neuronal nuclei (NeuN

Imaging
Culture plates were scanned using the Opera LX high content screening system (PerkinElmer). For each condition, three wells for astrocyte monolayers or five to six wells for astrocyte-forebrain co-cultures were analyzed. For cell counts, 14 images (20x objective) were captured per well. For quantitative analysis of neurite densities and MBP, 30 images (40x objective) were captured per well. For quantitative analysis of synapses 50 images (40x objective) were captured per well.

Quantitative analysis
Quantitative analysis was performed using Columbus 2.2 (PerkinElmer). Neurite (NF165, NF200, SMI31, or SMI32 for axons and MAP2 for dendrites) densities were given as area occupied by neurites relative to total area of the image (with or without correction for differences in number of NeuN + neurons). MBP was quantified as area occupied by MBP immunoreactivity relative to total area of the image. Synaptic density was calculated by counting synapses (VGLUT1 + or PSD95 + ) located on top of a dendrite or within a distance of 1.6 lm from a dendrite and dividing the number by total dendrite length per image. Co-localization of VGLUT1 + and PSD95 + was determined by searching for PSD95 + spots located on top of VGLUT1 + spots or within a distance of 0.65 lm from VGLUT1 + spots. SMI31-or SMI32-positive neurites were quantified when co-localized with NF200-or MAP2-positive neurites and given as total length in lm per well or as total length ratio relative to NF200 or MAP2. Percentage of microglia present in the astrocyte monolayer was determined by counting CD11b-positive cells relative to the number of nuclei (oligodendrocytes or neurons were not detected).

Electron microscopy
Previously, we reported on electron microscopy (EM) analysis of corpus callosum of 7-month-old wild-type, 2b4 ho and 2b5 ho mice and 4-month-old 2b4/2b5 heho mice, because the latter do not live up to 7 months. 23 Here, we additionally analyzed wild-type and 2b5 ho mouse corpus callosum at 2 and 4 months (two animals per group). Mouse brains were drop-fixed in 2% glutaraldehyde and 4% PFA in 0.1 mol/L sodium cacodylate buffer (pH 7.4). Human EM analysis included formalin-fixed corpus callosum tissue of a female 10-year-old control subject that died due to a brainstem tumor and a female 10-year-old patient diagnosed with classical VWM. Corpus callosum was dissected, postfixed in 1% osmium tetroxide and 1.5% potassium ferricyanide in 0.1 mol/L sodium cacodylate buffer, dehydrated and embedded in epoxy resin. Sagittally cut ultrathin sections were contrasted with uranyl acetate and lead citrate and analyzed using a FEI Tecnai 12 electron microscope. A peer researcher renamed the images in order to guarantee blinded quantifications by the assessor. Axonal diameters and myelin sheaths thicknesses were measured for at least 400 axons per genotype using ImageJ and G-ratios, defined as axon diameter/total axon + myelin diameter, were calculated. As most axons are not completely circular, the shortest diameter was measured. In addition, percentages of unmyelinated axons and total number of axons were quantified for at least 10 fields of view per genotype.

Golgi staining procedure
Golgi stain was performed on fresh 4-month-old mouse brains (two wild-type and two 2b5 ho mice) using the FD Rapid GolgiStain Kit (FD Neurotechnologies, Inc.) according to the manufacturer's instructions. Tissue sections were cut at 150 lm, stained, and coverslipped with DePeX (Serva), after which images were acquired on a confocal microscope (Zeiss LSM510) with a 10x objective (numerical aperture 0.3, 0.7x mechanical zoom) or a 40x oil objective (numerical aperture 1.3, 1x or 2.5x mechanical zoom), as z stacks with a 1-lm interval. Images were analyzed with the freely available RECONSTRUCT software (http://synapses.clm.utexas.edu). 32 Spine densities were determined along primary apical dendrites of layer V pyramidal neurons, starting quantification at a distance of 25 lm from the cell body.

Statistical analysis
Mouse EM data were tested for median differences using Mann-Whitney U test or Kruskal-Wallis test followed by post hoc Mann-Whitney U test, as assumptions for parametric tests were not met.
Independent co-culture experiments were performed at least four times with at least three replicates per co-culture condition. To analyze the data of all experiments simultaneously, we ran a multi-level analysis with experiment as clustering variable. Astrocyte genotype, forebrain genotype, and their interaction term were included as fixed effects. The interaction term between astrocyte and forebrain mixed cells was removed from the models in case this term was not significant. To accommodate variation across experiments in a specific outcome parameter, and variation in the effects of astrocyte genotype and forebrain genotype, random effects for these parameters were included in the model as nuisance parameters. Data are presented in multiple plots comparing two possible combinations of astrocyte-forebrain (wild-type vs. 2b5 ho cells) co-cultures, with each data point representing the mean of an individually performed experiment. Spine data were analyzed using an independent Student's t test.
All data were analyzed using two-sided tests. As effect size, Pearson's correlation coefficient r was used, with values of .10, .30, and .50 denoting a small, medium or large effect, respectively. A Bonferroni correction of the a was applied (standard a = 0.05) when performing multiple  Whereas axonal diameter and myelin sheath thickness were normal or close to normal in 2-and 4-month-old 2b5 ho mice, axonal diameter was smaller in 7-month-old 2b5 ho than in wild-type mice (wild-type median 0.61 vs. 2b5 ho median 0.33, Z = À11.05, ****P < 0.0001, r = 0.46), while myelin sheath thickness remained close to normal. In the 2b4/2b5 heho mice, myelin sheaths were thicker than normal (wild-type median 0.64 vs. 2b4/ 2b5 heho median 0.75, Z = À3.91, ****P < 0.0001, r = 0.14). In the 2b4 ho mice, the axons were smaller compared to wild-type mice (wild-type median 0.61 vs. 2b4 ho median 0.49, Z = À2.89, #P = 0.0039, r = 0.11; which is a borderline significant trend with a Bonferroni-corrected P value of 0.0033 for all 15 comparisons performed in A and B). Slopes of correlations between myelin sheath thickness and axonal diameter were similar between wild-type mice and 2b4 ho and 2b5 ho mutants, but slightly higher in the 4-month-old 2b4/2b5 heho mice. (B) G-ratio's were comparable in 2and 4-month-old wild-type and 2b5 ho mice and in 7-month-old wild-type and 2b4 ho mice. Aberrant G-ratios were detected for 4-month-old 2b4/ 2b5 heho mice (wild-type median 0.76 vs. 2b4/2b5 heho median 0.72, Z = À5.78, ****P < 0.0001, r = 0.20) and 7-month-old 2b5 ho mice (wild-type median 0.78 vs. 2b5 ho median 0.66, Z = -11.68, ***P < 0.001, r = 0.49). (C) Wild-type mice showed a clear shift to thicker myelinated axons from 2 to 4, to 7 months of age, whereas the 2b5 ho mice showed a shift to thicker axons from 2 to 4 months but shifted back to smaller axons at 7 months, comparable as the 2-month-old mice. (A, B) Mann-Whitney U test.
comparisons. Normality tests and multi-level analyses were performed using IBM SPSS Statistics 22. All other statistical analysis and plots were carried out and prepared using Prism 6.0 (GraphPad Software, Inc).

Study approval
Mouse brain tissue was obtained in strict compliance with the animal welfare policies of the Dutch government and approved by the IACUC of the VU University of Amsterdam. Written informed consent was obtained according to the Declaration of Helsinki and was provided by patients or their parents for the collection and use of patients' samples. The Medical Ethics committee of the VU University Medical Centre approved the procedures.

Results
Secondary axonal atrophy in VWM mice EM analysis of the corpus callosum of mice of different ages was performed to investigate the onset and development of possible abnormalities in axon size and axon-myelin proportion (Fig. 1). There was an impact of disease severity on axon diameter and axon diameter-myelin thickness ratio. In clinically presymptomatic 2b5 ho mice at 2 and 4 months, no clear differences were seen compared to wild-type mice, although myelin sheaths tended to be slightly thinner ( Fig. 2A, B). By contrast, in clinically symptomatic 2b5 ho mice at 7 months, clear differences were seen. While myelin sheaths remained on average slightly thinner than in wild-type mice, axons had become disproportionately thin for the myelin sheath thickness, resulting in a lower G-ratio than in wild-type mice, especially for thinner axons (Figs. 1 and 2A, B). Interestingly, a few myelin sheaths were much thicker than normal. In the more severely affected 2b4/2b5 heho mice, abnormalities were already present at 4 months, which is end-stage for these mutants. Myelin sheaths were thicker and axons were slightly thinner than in 4-month-old wildtype mice, resulting in a decreased G-ratio ( Fig. 2A, B). Strikingly, again a few myelin sheaths were seen that were much thicker than normal. In the less severely affected 2b4 ho mice, abnormalities were less obvious; myelin sheaths were close to normal, but axons were relatively small, although with a striking variation in diameter and presence of several very thick axons. Like wild-type mice, the G-ratio was similar for different axonal sizes. A few myelin sheaths were thicker than normal, but less so than in 4-month-old 2b4/2b5 heho mice and 7-month-old 2b5 ho mice.
Regarding the diameter distribution of myelinated axons at different ages, an increase in axonal size in both 2b5 ho mutant and wild-type mice occurred between 2 and 4 months, with a further increase in 7-month-old wild-type mice, while axonal diameter distribution in 7-month-old 2b5 ho mice had reversed to that of 2-month-old mice, indicating secondary axonal atrophy (Fig. 2C). Regarding variation in myelin sheath thickness in wildtype and 2b5 ho mice at different ages, 2b5 ho mice had slightly higher frequencies of very thin sheaths at 2 and 4 months and a greater variation in thickness at 7 months (data not shown).

Thin myelin sheaths and axons in VWM patients
EM analysis of human corpus callosum clearly showed thinner myelin sheaths in the VWM patient than in the control, but because of postmortem artifacts and long-term storage in formalin before being processed for EM, no reliable quantification and statistical analysis could be performed (Fig. 3). Histochemical and immunohistochemical analysis of patients brain tissue revealed that axons were normal where tissue damage was mild (mild tissue rarefaction), but were thin where tissue damage was at least moderate (severe tissue rarefaction to cavitation, data not shown).
High numbers of thin, unmyelinated axons and increased axonal density in 2b5 ho mouse corpus callosum; increased axonal density in primary co-cultures EM analyses of 7-month-old wild-type, 2b4 ho , 2b5 ho and 4-month-old 2b4/2b5 heho mice revealed presence of many thin, unmyelinated axons in the mutants' corpus callosum, with the highest numbers in the double-mutants (Fig. 4A). Additional EM analyses of younger animals showed that higher percentages of thin unmyelinated axons were already present from 2 months onward, although less high than at 4 and 7 months (Fig. 4B). These thin axons were associated with a higher axonal density at 4 and 7 months (Fig. 4C).
In order to further explore the development of axonal abnormalities, we prepared primary myelinating cell cultures consisting of a layer of mouse forebrain mixed cells, including neurons, oligodendrocytes, microglia, and a few astrocytes, plated on a monolayer of mouse forebrainderived astrocytes, cultured together for 4 weeks. The astrocyte monolayers contained~6.5% or~3.1% microglia when derived from wild-type or 2b5 ho mice, respectively (data not shown). Co-culture data of all experiments were analyzed simultaneously with multilevel analysis. When cultured with wild-type mixed forebrain cells, a monolayer of 2b5 ho astrocytes suppressed total MBP amounts compared to a monolayer of wildtype astrocytes, (Fig. 5A, t(5.03) = À2.73, P < 0.05, r = 0.77) and in most cultures, the percentage of Olig2positive cells expressing MBP, although the latter was Figure 5. Higher axonal density in VWM astrocyte-based co-culture. (A) MBP amounts (t(5.03) = -2.73, P < 0.05, r = 0.77) and in most cultures (B) percentages of MBP-expressing Olig2 + cell numbers (although overall not significant, t(4.03) = À1.69, P = 0.17, r = 0.64) were lower when wild-type forebrain mixed cells were cultured on 2b5 ho astrocytes than if cultured on wild-type astrocytes, while having no effect on (C) the total number of Olig2 + cells (P = 0.68). (D) Co-culture of wild-type forebrain cells on wild-type versus 2b5 ho astrocytes stained for axons (NF200, red), dendrites (MAP2, green), and neuronal cell bodies (NeuN, cyan). For the sake of clarity, the astrocytes themselves are not shown. Scale bar: 20 lm. A higher axonal density was obtained when wild-type forebrain cells (t(7.07) = 3.86, **P < 0.01, r = .82, (E) or 2b5 ho forebrain cells (t (5.07) = 2.52, P = .052, r = 0.75, not shown) were cultured on 2b5 ho astrocytes than on wild-type astrocytes, which was partly related to variance in neuronal cell numbers (F, G). (H) A similar effect on axonal density was detected in cultures consisting of two layers of 2b5 ho cells in comparison to wild-type cells (t(5.10) = 3.92, *P < 0.05, r = 0.89), associated with a higher neuronal cell number (t(49.08) = 3.28, **P < 0.01, r = 0.42, (I, J). No effect on axonal density was detected when 2b5 ho forebrain cells in comparison to wild-type forebrain cells were cultured on either wild-type astrocytes (K-M) or 2b5 ho astrocytes (not shown). (E-M) The different plots present data of the same five to seven individual experiments. Each data point represents the mean of an individually performed experiment, for the specified co-culture combination. Statistics were obtained by multi-level analysis on the raw data of all experiments combined. Astr = astrocyte monolayer; Fb cells = forebrain cells. overall not significant (Fig. 5B, t(4.03) = À1.69, P = 0.17, r = 0.64), while having no effect on the total number of Olig2 + cells (Fig. 5C, P = 0.68).
Regarding the effect of 2b5 ho astrocytes compared to wild-type astrocytes on axonal density, we found a significant main fixed effect for astrocyte (t(6.88) = 3.48, P < .01, r = 0.80), but no main fixed effect for forebrain mixed cells (t(4.92) = 1.31, ns, r = 0.51), indicating that the genotype of the astrocyte monolayer does affect axonal density, but the genotype of the forebrain mixed cells on top does not. Further analysis demonstrated that the 2b5 ho astrocyte monolayers promoted a higher axonal density than wild-type astrocyte monolayers in wild-type forebrain cells (Fig. 5D, E; t(7.07) = 3.86, P < 0.01, r = 0.82) without changing neuronal cell numbers (Fig. 5F, G). The difference in the effect of 2b5 ho astrocytes compared to wild-type astrocytes on axonal density for 2b5 ho forebrain cells was smaller (data not shown; borderline significance, t(5.07) = 2.52, P = .052, r = 0.75). Comparing cultures in which both the astrocyte and forebrain layers were either 2b5 ho mutant or wild-type showed that the effect of mutant astrocytes compared to wild-type astrocytes on axonal density was similar to the effects observed in wild-type forebrain cells ( Fig. 5H; t (5.10) = 3.92, P = 0.01, r = 0.89, vs. Fig. 5E), indicating lack of a significant additional effect of the mutant forebrain cells on axonal density. Neuronal cell numbers in cultures with both layers consisting of 2b5 ho cells were higher than when both layers consisting of wild-type cells (t(49.08) = 3.28, P < 0.01, r = 0.42, Fig. 5I, J), indicating some effect of the mutant astrocytes combined with the mutant forebrain cells on neuronal cell number. When cultured on wild-type astrocytes, 2b5 ho forebrain cells had no effects on axonal density or neuronal cell number compared to wild-type forebrain cells (Fig. 5K-M) and neither did they have an effect when cultured on 2b5 ho astrocytes (data not shown).
Regarding effects on dendritic density, neuronal cell body size and on the ratio between axonal and dendritic density, no significant differences were detected with these combinations of wild-type or 2b5 ho astrocytes and forebrain cells (Fig. 6 and data not shown).

Evidence of mild axonal damage in VWM patient white matter
In order to assess whether axonal swellings and spheroids primarily occur in regions with severe tissue damage, we microscopically examined brain tissue of VWM patients for the presence of axonal swellings and spheroids using immunohistochemistry. In most patients, only a few scattered swellings and spheroids were detected (Fig. 7A). Also in severely damaged tissue only a few spheroids were found (Fig. 7B), the presence of which was not dependent on brain region (Fig. 7C). Of note was the presence of many spheroids in the occipital lobe of one of the oldest VWM patients (37 years at demise, Fig. 7D), while tissue structure and myelin content (not shown) were relatively preserved. No obvious signs of impaired axonal cytoskeletal architecture (SMI31 vs. SMI32) were detected in patient tissue and co-culture experiments (data not shown). Figure 6. No significant differences in dendritic density and neuronal cell body size between co-cultures consisting of 2b5 ho versus wild-type mouse-derived cells. No effect of 2b5 ho in comparison to wild-type astrocytes on neuronal cell body size was detected when wild-type forebrain cells (A-C) or 2b5 ho forebrain cells (not shown) were cultured on top. Also two layers of 2b5 ho versus wild-type cells (D-F) or 2b5 ho forebrain cells on either (C) wild-type astrocytes or 2b5 ho astrocytes (not shown) did not give a difference in dendritic density or neuronal cell body size. (A-I) The different plots present data of the same five to seven individual experiments, the same experiments as plotted in Fig. 4. Each data point represents the mean of an individually performed experiment, for the specified co-culture combination. Statistics were obtained by multi-level analysis on the raw data of all experiments combined. Astr = astrocyte monolayer; Fb cells = forebrain cells. Unaffected synapse or spine density in primary co-cultures or along primary apical dendrites of layer V pyramidal neurons Synaptic densities were quantified in co-cultures of various combinations of wild-type and 2b5 ho mouse-derived cells (Fig. 8A-F). No difference in synaptic density was found when wild-type forebrain mixed cells (P = 0.17, Fig. 8D) or 2b5 ho forebrain mixed cells (P = 0.40, not shown) were cultured on a monolayer of 2b5 ho astrocytes compared to wild-type astrocytes. Also no significant effect of 2b5 ho versus wild-type forebrain cells on synaptic density was found with either wild-type astrocytes (P = 0.09, Fig. 8E) or 2b5 ho astrocytes (P = 0.20, data not shown). A trend for a lower synaptic density was found when both layers consisted of 2b5 ho cells compared to two layers of wild-type cells (t(5.09) = À2.334, P = 0.07, r = 0.72; Fig. 8F).
In Golgi stains of fresh 4-month-old mouse brains ( Fig. 8G-I), no difference in spine density was detected along apical dendrites of wild-type versus 2b5 ho layer V pyramidal neurons (Fig. 8I).

Discussion
VWM is a devastating leukodystrophy, for which the underlying cellular mechanisms have yet to be unraveled. The current view is that astrocytes and oligodendrocytes are selectively affected, and that astrocyte pathology drives oligodendrocyte pathology. 23 We previously developed mutant VWM mice that are representative of the human disease. 23 Here, we studied axonal abnormalities in VWM Eif2b4-and Eif2b5-mutant mice with different disease severities and of different ages, in order to assess whether and when axonal abnormalities occur. In addition, we used primary astrocyte-forebrain co-cultures based on brain cells of the Eif2b5 mice to assess whether axonal abnormalities are primary or secondary to abnormality of VWM astrocytes. We verified findings in brain tissue of patients.
In 2b5 ho mice, average axonal diameter and relative myelin sheath thickness and their variation are initially normal, although myelin sheaths tend to be slightly thinner from early on. It is only at 7 months that axons are disproportionally small and myelin sheaths are still slightly thinner than normal, but thicker than expected for the axonal diameter, reflected by a lower G-ratio, especially for the smaller axonal diameters. The distribution of axonal diameters in 2b5 ho mice is close to normal at 2 and 4 months with a normal shift to greater axonal diameters at 4 months. However, the distribution of axonal diameters at 7 months has returned to that at 2 months, while in wild-type mice, a further shift to larger diameters has occurred. In the more severely affected 2b4/2b5 heho mice, deviating results for axonal diameter, relative myelin sheath thickness, and G-ratio are already present at 4 months. In the less severely affected 2b4 ho mice, differences from wild-type mice at the age of 7 months are less clear than in 2b5 ho mice of the same age. Overall, our results indicate that axons and relative myelin sheath thickness are initially normal or close to normal and only secondarily axonal atrophy occurs, dependent on disease severity. Previously, a different Eif2b5 mutant mouse model was reported to display a higher proportion of thin axons in the internal capsule associated with thinner myelin sheaths and a normal G-ratio at 3 weeks. 33 Axonal diameter and myelin sheath thickness at later ages were not reported, but it was demonstrated that in the cerebral peduncle of 15-monthold mutants an increased proportion of axons had no or thinner myelin than of wild-type mice.
The mouse findings were in part confirmed in VWM patient tissue. Notably, patient tissue was obtained at autopsy and therefore at end-stage disease, whereas mice were sacrificed at the time of appearance of clinical symptoms. EM analysis of the corpus callosum of a VWM patient suggested that myelin sheaths are thinner than in an age-matched control subject, but, as explained above, no definitive quantification of myelin sheath thickness or axonal diameter could be performed. However, examination of multiple patients' white matter regions overall demonstrated that axons were normal where tissue damage was mild, but were thin where tissue damage was moderate to severe, in line with the findings in the VWM mice. Thin axons in human brain tissue have been described before. 6 Taken together, these data indicate that axonal morphological aberrancies are related to the severity of the white matter disease.
A new finding is the presence of numerous thin, unmyelinated axons in the corpus callosum of mutant mice, not present in similar numbers in wild-type mice, resulting in increased axonal density. The highest percentage was found in the most severely affected 2b4/2b5 heho mice, suggesting that the density of thin unmyelinated axons also correlates with disease severity. Our mouse astrocyte-forebrain co-culture model provided us with the opportunity to assess if axonal density is related to the influence of mutant astrocytes on axons. Interestingly, in 2b5 ho mouse-derived cell cultures axonal density is also increased compared to wild-type cultures. A layer of 2b5 ho astrocytes suffices to achieve this effect in wild-type or 2b5 ho forebrain cells; a layer of 2b5 ho astrocytes cultured with 2b5 ho forebrain cells does not have an additional effect. Notably, a layer of 2b5 ho forebrain cells on wild-type astrocytes is associated with a normal axonal density. These findings indicate that the effect is not intrinsic to axons or neurons, but mediated by 2b5 ho astrocytes, which promote higher axonal density. These data imply that axons are not primarily affected in VWM, but atrophy secondarily due to astrocytes or astrocytesecreted factors.
Another new finding is the observation of some very large axons in the 7-month-old 2b4 ho mice and some very thick myelin sheaths in the 4-month-old 2b4/2b5 heho mice and 7-month-old 2b4 ho and 2b5 ho mice, associated with a very low G-ratio. Apparently the strict relationship between axon diameter and myelin sheath thickness is disrupted in more than one way. The cause of this remains unclear.
Previously, we showed that astrocyte pathology is a first sign of the disease in VWM mice and occurs before myelin pathology: we observed immaturity of astrocytes at postnatal day 14, followed by lower than normal MBP expression at day 21 in the same 2b5 ho mouse model. 23 In co-cultures of different composition, we demonstrated that oligodendrocyte maturation defect and lack of MBP production are induced by VWM astrocytes and not intrinsic to VWM oligodendrocytes. 23 In thist study, we confirmed that a layer of 2b5 ho astrocytes in astrocyteforebrain co-cultures inhibits total MBP production and the percentage of MBP-expressing Olig2 + cells.
All available evidence indicates that VWM astrocytes are primarily affected and axonal pathology is also secondary. It is an open question whether the process of axonal dystrophy is directly caused by the astrocytes through, for example, lack of energy supply or secretion of a toxic factor, or mediated through the impact of astrocytes on oligodendrocytes and their ability to provide metabolic support to axons. 34,35 The effect of astrocytes on axonal density may be related to immaturity of VWM astrocytes, which better promote axonal growth or regeneration than mature astrocytes, while having no differential effect on dendrite growth. [36][37][38][39] It has been postulated that astrocytes provide general support for process growth through secreted factors and that the differential effect of mature versus immature astrocytes on axonal growth, but not on dendritic growth, is due to cell-cell contact. 40 Axonal swellings and spheroids, evidence of disturbed axonal transport, are rare in VWM patient tissue. Most patients have only a few scattered swellings or spheroids throughout the brain white matter, even in the presence of severe white matter damage. The current observation of the highest number of spheroids in one of our oldest patients, and the previous observation of spheroids in older but not in younger 2b5 ho mice suggests that axonal transport problems develop over time. 23 The mechanism is unclear. We found no clear signs of impaired axonal cytoskeletal architecture in patient tissue and co-culture experiments. The older patient, who had a high number of spheroids, also had relatively intact myelin, indicating that spheroids are not caused by absence of myelin. Anyhow, disturbed axonal transport does not appear to be a central problem in VWM.
It is likely that the clinical features in VWM are at least in part caused by axonal dysfunction. In our 2b5 ho mice, clinical signs arise around the age of 5 months. Interestingly, the shift from normal-sized axons to disproportionally thin axons occurs between the ages of 4 and 7 months. These abnormalities potentially lead to a reduction in signal conduction velocity, as observed in VWM patients, which correlates more closely to axon diameter than fiber (axon + myelin) diameter. 41 To conclude, current data support the concept that astrocytic dysfunction drives VWM pathology, affecting not only oligodendrocyte mature functions but also the trophic condition of axons. A very important finding is that axons and relative myelin sheath thickness are initially normal or close to normal, indicating a window of opportunity for treatment. It may be possible to prevent axonal morphological changes when interventions are undertaken in time.


s2fieldsofstudy: Biology Medicine

text:
Evaluation of serum hepcidin as a biochemical marker in diagnosis of anemia of chronic disease

Anemia is a serious public health problem. It affects two billion people worldwide, particularly infants and young children mainly in developing countries where its etiology is multifactorial). Anemia in infancy is generally associated with impaired cognitive and behavioral development, impaired oxygen transport and a poorer prognosis in the context of many chronic diseases, including HIV iron bindings or chronic infections. Hepcidin, is a peptide hormone that is synthesized and regulated in response to iron status and the innate immune system, it evolves as the master regulator of iron metabolism, linking iron homeostasis, inflammation, infection and anemia. The molecular control of hepcidin is a part of the innate immune response to pathogens and is stimulated by IL-6, IL-22, type I interferon’s, toll-like receptor (TLR) ligands, and the endoplasmic reticulum ABSTRACT

INTRODUCTION
Anemia is a serious public health problem. It affects two billion people worldwide, particularly infants and young children mainly in developing countries where its etiology is multifactorial). [1][2][3] Anemia in infancy is generally associated with impaired cognitive and behavioral development, impaired oxygen transport and a poorer prognosis in the context of many chronic diseases, including HIV iron bindings or chronic infections. [4][5][6][7] Hepcidin, is a peptide hormone that is synthesized and regulated in response to iron status and the innate immune system, it evolves as the master regulator of iron metabolism, linking iron homeostasis, inflammation, infection and anemia. The molecular control of hepcidin is a part of the innate immune response to pathogens and is stimulated by IL-6, IL-22, type I interferon's, toll-like receptor (TLR) ligands, and the endoplasmic reticulum stress response. 8 Hepcidin synthesis is reportedly stimulated by elevated plasma iron concentration, infection and/or inflammation, and is suppressed in conditions that demand increased serum iron, such as increased or ineffective erythropoiesis, hypoxia, anemia and also iron deficiency. [9][10][11][12] A number of hepcidin studies have been conducted in adults. [13][14][15] Ganz et al, created a reference for plasma hepcidin concentration in adult men and women and reported a 5% to 95% range of 29 to 254ng/mL with a median 112ng/mL in men (n=65) and 17 to 286ng/mL with a median 65ng/mL in women (n=49) using the first competitive ELISA. 13 Grebenchtchikov et al, developed a new RIA (CV 4-6% range) and reported significantly higher median hepcidin concentration in men compared to women. 15 However, a study using SELDI-TOF MS did not find a significant difference in hepcidin concentrations between men and women. 14 Galesloot et al, extended these findings using a similar competitive ELISA and established age-and sex-stratified reference ranges (median, 2.5th and 97.5th percentiles) for serum hepcidin concentration using a population-based sample from the Netherlands (n = 2998). 16,17 Participants who were pregnant, had alanine aminotransferase (ALT) >50U/L, CRP >10mg/L, estimated glomerular filtration rate <60mL/min/1.73 m2, using iron supplements, anemic, or had a BMI > 30kg/m2 were not considered to be eligible for the study, however no strict definition of 'healthy' was employed. 18 Grebenchtchikov et al, and Galesloot et al, both reported diurnal variation in hepcidin levels, however their results are inconsistent. Galesloot et al, reported lower hepcidin values from blood samples obtained in the morning (before 12pm) compared to blood samples obtained between 12 and 5 pm in both men and women. In contrast median hepcidin levels were 1.83 and 1.70 times higher at 9 am compared to 4 pm in men and women respectively in a study by Grebenchtchikov et al. 15,16 Hepcidin concentrations were constant over age in men median (2.5th and 97.5 th percentiles): 7.8nM (0.6 -23.3nM) but were higher in postmenopausal compared to premenopausal women (4.1nM (0.4 -19.7nM) in women <55 years and 8.5nM (1.2-24.8nM) in women >55 years). 15 In another study, Galesoot et al, reported lower median hepcidin concentrations compared to Ganz et al, and found a less pronounced difference in hepcidin concentrations between men and women. These differences might be due to the fact that the 2 studies used different assays and women in the study by Galesoot et al, had a higher median age (55 years) compared to women in the study by Ganz et al (32.6 years). 13,16 Although the two studies used similar immunochemical assays, they used different antibodies highlighting the need for harmonizing hepcidin assays although the difference in absolute hepcidin values obtained by different assays precludes the comparison of hepcidin concentrations across different assays, among studies using the Ganz assay, hepcidin concentrations in infants are consistently lower compared to those in adults. These infant studies included cord blood samples from healthy term infants, neonates and preterm infants 35 days postnatal. 14,[19][20][21] In the present study, we determined the hepcidin to characterize normative plasma hepcidin concentrations in healthy non-anemic Indian subjects and to determine the independent effects of infection/ exposure and anemia on hepcidin concentrations. Understanding normative hepcidin levels in the selected populations will allow us to understand the hormonal regulatory mechanism that healthy subjects from developing anemia. Authors set out to measure plasma hepcidin concentrations and to characterize normative values in healthy and ACD anemia group. We used an algorithm developed of data to generate hepcidin consensus values as cut off to allow for harmonization with different hepcidin assays.

METHODS
This is a prospective analytical case control study. Patients attending the outpatient and inpatient department of the INDEX hospital were included during study period of two year from June 2014 to June 2016. Appropriate prior consent was taken from the patients and clinical details were recorded in a proforma. All investigations were done in the pathology and clinical biochemistry continue the para/ remove the tab. laboratory of INDEX Hospital, Indore. Sample for complete blood count and biomarkers analysis were collected, coded and processed on a SYSMEX X-800i auto analyzer for hematological parameters and on ELAN 30S ELISA plate reader for Serum Hepcidin, Ferritin, IL-6 levels analysis. A total of 86 patients were evaluated, those full filling the inclusion criteria and allocated in two groups according to their RBC's indices, peripheral smear examination and serum ferritin values.
Control (Non-anaemic) were those with Hb level (meanSD) 13.010.69 gm/dl and RBCs count (10 6 /µl; meanSD) 4.460.21 included within normal range to their respective reference age, sex and normal ferritin levels and a normal peripheral blood smear diagnosis. ACD group comprised of cases with Hb level (meanSD) 8.652.88gm/dl and RBCs count (10 6 /µl; meanSD) 3.541.14 lower than the normal range to their respective reference age and sex and a hypochromic picture on peripheral blood smear. The biomarkers levels for both groups were analyzed, studied and compared. Patients already on iron supplements, blood transfusion in recent months and below 5 years of age (to exclude congenital conditions like thalassemia, haemochromatosis) were excluded from the study. The obtained data was tabulated using MS Excel to create a master chart. The power of study was kept at 99% and level of significance (α) at 5%. "Analysis of Variance (ANOVA)" was done to calculate the p value to compare the difference of mean of the study groups together. Post Hoc Turkey's test" was also applied for comparison of difference of mean in two study groups. The p value was calculated for each parameter and p value <0.05 was considered to be significant. 95% CI was also calculated. "Pearson's Correlation Coefficient (r)" was determined to find out the correlation between serum hepcidin and serum ferritin, serum IL6 in studied groups. X-Y scatter plots and other graphical representation of data were done with appropriate plots and charts as needed depending on data type and distribution. AUC under ROC curves was calculated for serum hepcidin as a potential diagnostic test for detection of IDA in studied groups using Statistical Package for Social Sciences (SPSS) version 24.0 for windows and Medcalc Software.

RESULTS
The hematological parameters are summarized in Table 1 for group ACD, and normal. The patients had elevated WBC count as compared to normal group. The average meanSD value recorded for ACD and normal group were 11.485.78ng/mL, and 8.152.22ng/mL respectively. The results well commensurate with the findings reported by chang et al. 22 In this study, the serum ferritin measured in patients of normal group was 65.6716.76ng/mL while in case of ACD groups the serum ferritin level measured was elevated to 215.5015.9ng/mL which was significantly higher than other groups. The calculated P value suggested significant difference (P≤0.0001). The serum hepcidin measured in patients of normal group was 54.0615.46ng/mL while in case of ACD group the serum hepcidin level was elevated 98.3624.29ng/mL which was significantly higher compared to other groups. The calculated P Value suggested a significant difference (P ≤0.005). The serum IL-6 measured in patients of normal group was 10.532.91ng/mL while in of ACD group the serum IL-6 level was elevated to 115.8233.7ng/mL which was significantly different and highest among all the groups. The calculated P Value suggested a significant difference (P ≤0.005).

Receiver operating curve and scattered plot correlation curve
Iron deficiency anaemia is characterized by many abnormal laboratory features. Because none of these are unique, a small deviation from normal will detect most cases of iron deficiency (high sensitivity), but also falsely identify non-iron deficient subjects as being iron deficient (low specificity). On the other hand, a large deviation from normal will exclude most non-deficient patients (high specificity) but miss many iron deficient subjects (low sensitivity). The tradeoff shown graphically is so called receiver operating characteristic curve (ROC). These curves are constructed by plotting the sensitivity against the false positive rate (1-specificity) at various values of the analyte. The ROC curves for serum hepcidin as predictors of ACD was constructed using MedCalc software. Further, the correlation(s) between different biomarker(s) was assessed by plotting the bio exponents on X and Y axis: the scattered correlation curve so obtained was used to compare correlation coefficient and level of significance at 95% confidence level with defined confidence interval. In case of normal group, the scatter correlation curve revealed an excellent positive correlation between serum ferritin and hepcidin wherein for the sample size of 34 participants, correlation coefficient obtained on regression of curve was 0.9744, significance level P<0.0001, 95% confidence level interval 0.9489 to 0.9873. The values suggest positive correlation between the hepcidin and ferritin indicating if ferritin level may define the cut off point for anemia then hepcidin could equal and effectively be used for defining and determining ID.
Authors found a significant positive correlation between serum ferritin and hepcidin which is consistent with numerous reported studies. The serum hepcidin as well as ferritin being acute phase reactants of immune activity; are raised especially in chronic inflammatory disease hence in case ACD group the correlation was observed to be strongly positive with correlation coefficient -0.7984; significance level P <0.0001 at 95% confidence level 0.6719 to 0.8796 ( Figure 1). As per proposed mechanism by Nemeth et al, this could be due to the fact that IL-6 is necessary and sufficient for hepcidin expression induction establishing that iron regulatory peptide hepcidin plays a key role in iron metabolism. 24 The study demonstrated a positive correlation between serum hepcidin and IL-6. The correlation coefficient was 0.6359 with significance level P <0.0001 at 95% confidence interval r = 0.4392 to 0.7744 inflammatory cytokines IL-6 was able to strongly stimulate hepcidin expression and as consequence such indicator could be responsible for hypoferriemia accompanied with chronic inflammatory diseases, This is in accordance with the report of kato et al, Nemeth et al. 24,25 Figure 1: Receiver operating characteristic curves for serum hepcidin in ACD group.

DISCUSSION
Hepcidin a peptide and a regulator of iron metabolism is expressed in iron deficiency and also it has sensitive method of estimation i.e. immunoassay (ELISA based) was used as a marker and critically evaluated in relation to ferritin and IL-6 levels where in IL-6 being a specific cytokine expressed in chronic infection(s) inflammations. 26,27 In present study, authors observed statistically significant lower values of RBC count, Hb gm/dl, MCV, MCH, MCHC in ACD group then the normal group. In case of hepcidin when ROC curves and Pearson's scattered plot were made for ACD group; findings showed that ROC was maximum >0.869; with a sensitivity of 84.62% and specificity 94.12% while the confidence level was 95% with an interval of 0.779 to 0.932 in the case of ACD group. Further, the cutoff point determined was >72.93. Thus, the hepcidin level >72ng/mL and above is related to the ACD.

CONCLUSION
This is the pioneer study of its kind, (as per reported literature) conducted in India which is multi parameter centric and comprehensively conducted to explore the use of a biomarker i.e. hepcidin in detection and diagnosis in early stage anaemia. The available methods for iron deficiency anaemia diagnoses have their limitations as in the case of ferritin as marker it has excessive high values in chronic infections, malignancies, hepatitis, and hyperthyroidism. It is relatively less sensitive in detecting an early iron deficiency. Similarly, bone marrow aspiration is an invasive procedure, reportedly with nonpatient compliance; tedious; involves high skill and requires aseptic condition hence cannot frequently be used as a method of choice for the detection of iron deficient anaemia. It was found that hepcidin is positively correlated with IL-6 in the case of ACD means as an infection persists the iron deficiency is resulted which coconstitute a part of innate immunity and hence IL-6 as indicative marker of infection gets up regulated which correlate well with hepcidin expression (>72.93).