Minocycline Alleviate A Rat Model Of Gulf War Illness Via Altering Gut Microbiome, Attenuating Neuroin ammation And Enhancing Hippocampal Neurogenesis


 Background: Accumulating evidence suggests that deficits in neurogenesis, chronic inflammation and gut microbiome dysregulation contribute to the pathophysiology of Gulf War Illness (GWI). Minocycline has been demonstrated to be a potent neuroprotective agent and could regulate neuroinflammation. The present study intended to investigate whether treatment of minocycline maintain better cognition and mood function in a rat model of GWI and the potential mechanism. Methods: Rats received 28 days of GWI-related chemical exposure and restraint stress, along with daily minocycline or vehicle treatment. Cognitive and mood function, neuroinflammation, neurogenesis and gut microbiota were detected.Results: We found that minocycline treatment induced better cognitive and mood function in a GWI rat model, as indicated by open-field test, elevated plus maze test, novel object recognition test and forced swim test. Moreover, minocycline treatment reversed the altered gut microbiome, neuroinflammation and the decreased hippocampal neurogenesis of rats with GWI. Conclusion: Taken together, our study indicated that minocycline treatment exerts better cognitive and mood function in GWI rat model, which is possible related to gut microbiota remodeling, restrained inflammation and enhanced hippocampal neurogenesis. These results may establish minocycline a potential prophylactic or therapeutic agent for the treatment of GWI.

regulation 13 . Importantly, the impairment of adult hippocampal neurogenesis contributes to several psychiatric disorders, such as GWI, addiction, depression, schizophrenia and autism spectrum disorder 10,14−16 . Brain imaging studies of GWI patients suggested that the scores on immediate and delayed verbal and visual retrieval decreased in GWI patients, and the hippocampal volume, especially the hippocampal head, was signi cantly smaller than that of the healthy civilian 17 . Additionally, accumulating evidence suggests that the level of neuroin ammation in the brain play a role in adult neurogenesis. Over activation of neuroin ammation can adversely affect cognitive and mood function either directly or indirectly by inhibiting hippocampal neurogenesis 18,19 . Meanwhile, over activation of neuroin ammation have been identi ed in animal models of GWI using exposures to several chemical combinations 20 . For GWI patients, the levels of multiple proin ammatory biomarkers in the blood also increased signi cantly 21 . More importantly, enhanced neurogenesis, and alleviation of neuroin ammation by curcumin treatment could leads to better cognitive and mood function in GWI rats 22 . Taken together, the results of these studies have indicated that over activation of neuroin ammation and impaired neurogenesis in the adult hippocampus might be the key biological processes involved in GWI pathophysiology and may as the potential therapeutic targets for treating GWI.
Minocycline, as a long-acting tetracycline agent, has a profound neuroprotective effect associated with the improvement of cognitive function, mood regulation and neurogenesis during neuroin ammatory conditions. Several studies have reported that minocycline was bene cial in neurological models such as schizophrenia, depression, ischemic stroke, and Parkinson's disease [23][24][25][26] . However, the role of minocycline in promoting hippocampal neurogenesis and alleviating of neuroin ammation, including potential cognitive and mood enhancing properties in GWI, remains unclear. We here aimed to evaluate the effects of minocycline treatment on neuroin ammation and neurogenesis in the hippocampus in the rat model of GWI. We correlated these data with the effects of minocycline treatment on diminished cognitive and mood function of GWI rats.

Animals and groups
Adult male Sprague-Dawley rats (9 weeks old) were placed in pairs in the plexiglass cage in a temperature-controlled room (23-25 °C) with humidity 50-60 %, a 12-h light/12-h dark cycle and ad libitum access to food and water. The experiments were approved by the Animal Care and Use Committee of the General Hospital of Northern Theater Command and was in consistence with the principles outlined in the National Institutes of Health Guide. All efforts were made to minimize the number of animals used at each step. Rats were randomly divided into the following ve groups: Control group, GWI group, and GWI + Mino group.

Exposure of rats to PB, Permethrin and restrained-stress
The pyridostigmine bromide (PB) was purchased from Sigma (Burlington, MA, USA) and was administered by oral gavage at a dose of 1.3 mg/kg for 28 d (in 500 μL water). The rats exposed to chemicals DEET (in 200 μL 70% alcohol, 40 mg/kg) and PM (in 200 μL 70% alcohol, 0.13 mg/kg) over shaved skin areas located on the back of the neck and the upper thoracic region for 28 consecutive days.
The doses of PB, PM, and DEET were chosen according to previous reports. 10 Moreover, 5 min of daily restraint stress was conducted for 28 days by a rat restrainer, as previously reported. 27 2.3 Administration of minocycline As Figure 1 show that rats received PB, Permethrin and restrained-stress exposure and with normal saline intraperitoneal or minocycline (Sigma) dissolved in sterile saline, at dose of 40 mg/kg/day (minocycline) by intraperitoneal administration for 28 days.

BrdU labeling
Bromodeoxyuridine (BrdU; Sigma-Aldrich) was dissolved in sterile saline. In order to assess cell proliferation in the DG, rats were received BrdU at 50 mg/kg body weight dose intraperitoneally, for a total of 3 injections in 3 days, and were then sacri ced 24 h after the last BrdU injection. In order to analyze neurogenesis in the DG, mice were sacri ced 28 days after the rst BrdU injection, and BrdU positive cells as well as BrdU positive cells colabeled with doublecortin (DCX) were quanti ed.

Behavioral experiments
After GWI model established and levo oxacin, minocycline treatment, the behavioral tests were performed. Before each test, rats were acclimatized to the experimental room for at least 30 min. The behavioral tests were performed in the following order: open eld test, the elevated plus maze test, novel object recognition test and forced swim test.

Open eld test
The open-eld test was performed according to previous methods 28 , using a video camera secured to the top of the apparatus and analyzed using Ethovision 11.0 (Noldus). Rats were placed in the center of the open-eld box and activity was recorded for a period of 10 min. The distance and duration of time in the central area were recorded. After each test, the apparatus was wiped with alcohol and water between tests to eliminate any smell.

The elevated plus maze test
The elevated plus maze test was performed to measure anxiety-like behaviors, consisted of two open arms and two closed arms. According to a previous report 27 , the rat was initially placed in the center area facing an open arm. The duration of time for the rat to enter any of the 4 arms was recorded when all 4 paws crossed from the central region into an arm during the 10 min testing period. The duration of time spent in the open arms and the number of total arm entries were used for calculation of the extent of anxiety-like behavior in each rat.

Novel object recognition test
The novel object recognition test paradigm consisted of mainly two phases. In the adaptive phase, two identical cylindrical objects (A object) are placed on both sides of the test box, place the rat in the box for 3 minutes, and let the rat rest for 15 minutes. During the test phase, one of the cylindrical objects was replaced by a cube box (B object). The length of the bottom of the cube box is the same as the previous cylindrical object. The diameter of the cylindrical box is the same, then put the rat into the test box and let it explore for 3 minutes. The duration of the rat explores new object (B object) and old object (A object) were recorded and analyzed by Ethovision 11.0 (Noldus). As the distance between the tip of nose and object is less than 2 cm, de nite as exploration time. Exploration preference index = time for mice to explore new objects (B) / total time for mice to explore all objects (A + B) * 100%.

Forced swim test
Forced swim test is widely used for measurement of depression-like behavior in rodents 29 . Brie y, mice were individually placed for 6 min in beakers (height: 46 cm, diameter: 30 cm) containing 30 cm height of water (25 °C) for 6 min. During this time the rat adopts an immobile posture to keep its head above water, characterized by motionless oating and the cessation of struggling. The latency to adopt this posture and total time spent immobile during the last 4 min of the 6 min test were recorded. Increased duration of immobility indicated a state of helplessness.

Immunohistochemistry and immuno-uorescence
Immunohistochemistry was conducted according to previous method 28  Finally, sections were analyzed under a Lycra microscope.

Quantitative RT-PCR
Total RNA was extracted using the TRIZOL reagent (Sigma Aldrich) according to the manufacturer's instructions. And then cDNA was generated using the PrimeScript®RT Reagent Kit (Takara Bio Inc) and qPCR was performed in triplicate using 96 wells PCR plates and YBR Green qPCR Mix (Takara Bio Inc., Shiga, Japan) with a CFX96 Real-time PCR System (Bio-Rad) according to manufacturer's instructions. Cq values were normalized to GAPDH and were calculated using the 2−ΔΔC(t) method as described previously 30 .

ELISA
The concentration of rat IL-10 (Wuhan USCN) and IL-17 (Wuhan USCN) in the hippocampus and serum were measured by their respective ELISA kits according to the manufacturers' instructions. The results are expressed as picograms milligram (pg/mg) or picograms permilliliter (pg/ml).

Western blotting
The whole hippocampal tissues were homogenized in the RIPA buffer (Beyotime Institute of Biotechnology) and spun down at 13,000 rpm for 10 min, and then supernatants were collected. The protein concentration was determined with a BCA kit (Beyotime Institute of Biotechnology). The rest hippocampal homogenized lysates were performed at 100℃ for 10 min followed. The separated proteins were transferred onto a 12% SDS-polyacrylamide gel at 80 V for 120 min, then transferred to PVDF membranes. Blotting membranes were blocked in solution (5% nonfat dried milk powder dissolved in TBST buffer at room temperature for 3 h, and then washed three times. Next, membranes were incubated overnight at 4℃ with primary antibodies against TLR4 (1:1000, Santa Cruz Biotechnology), NF-κB p65 (1:2000, BD Biosciences), β-actin (1:2000, Cell CWBIO). The membranes were washed and incubated for 2 h with horseradish peroxidase-conjugated secondary antibody (1:1000, Santa Cruz Biotechnology). The bands were scanned and analyzed using a chemiluminescence system (Bio-RAD Laboratories Inc.).

Microbiome analysis
Fecal pellets and luminal contents were collected from the animals of each group after sacri ce, and then bacterial DNA from cecal contents was extracted using a DNeasy PowerSoil Kit (Qiagen) according to the manufacturer's instructions. 16s rRNA gene sequencing of fecal DNA samples was performed by NovaSeq sequencing platform. The V3-V4 region was ampli ed and sequencing was done using an Illumina HiSeq sequencing platform. Sequences from all samples were processed using QIIME (Knight and Caporaso labs) and then assigned by the RDP classi er (Michigan State University) against the Greengenes database.

Statistical analyses
All data are presented as the mean ± SEM and analyzed using SPSS 20.0 software (SPSS Inc., Chicago, IL, USA). Data analyses were performed using one-way ANOVAs and signi cant effects were evaluated with Tukey's post hoc tests. In the 16S rRNA gene sequencing analysis, a repeated-measure ANOVA was used to analyze the α-diversity of the microbiota to assess treatment-by-sequence interactions.
Statistically signi cance was set at p < 0.05.

Minocycline treatment alleviated neurobehavioral de cits in a GWI rat model
A timeline with the experimental design was shown in Figure 1A. As previous studies reported that animals with GWI exhibited increased levels of depressive-and anxiety-like behaviors 10 . Thus, we examined a cohort of rats using the open eld test and the elevated plus maze test to examine locomotor activity and anxiety-related behaviors. We found that minocycline treatment reverses decreased time in the central area ( Figure 1B) for GWI rats and decreased distance in the central area ( Figure 1C) in the open eld test. Meanwhile, the results from the elevated plus maze test showed that rats in GWI + Mino group displayed higher level in the percentage of time spent in open-arms ( Figure 1D) and percentage of open arm entries ( Figure 1E), compared with rats in GWI group. Moreover, as previously reported that decreased immobility time in the forced swim test is the index of antidepressant-like e cacy. We revealed that GWI rats showed increased immobility in the forced swim test, compared with control rats. While minocycline treatment reversed this effect signi cantly in GWI rats ( Figure 1F). These results indicated that minocycline treatment decreases depressive-and anxiety-like behaviors in GWI rats.
To test whether minocycline corrected recognition memory de cit in GWI rats, we performed the novel object recognition test. The results showed that GWI rats had a de cit in declarative recognition memory, whereas control rats exhibited a signi cant preference for the novel object. GWI rats with minocycline treatment for 28 days signi cantly improved recognition memory compared to that of the vehicleadministered GWI group ( Figure 1G). Meanwhile, the total exploratory time of all objects ( Figure 1H) or the total distance moved in the box ( Figure 1I) were comparable across the three groups, indicating that the results are not in uenced by potential changes in overall activity or lack of motivation. Thus, these ndings implied that minocycline treated GWI rats exhibited better recognition memory.

Minocycline treatment enhanced hippocampal neurogenesis in GWI rats
The hippocampal pathology of GWI typi ed by decreased neurogenesis, while enhanced hippocampus neurogenesis could alleviate GWI-related behaviors 10,22 . Therefore, we analyzed the proliferating cell population in the SGZ-GCL using the BrdU incorporation assay, which demonstrated that a reduced density of BrdU+ cells in GWI rats compared to control group. While minocycline treatment rescued the decreased BrdU+ cells in the SGZ-GCL of GWI rats signi cantly (Figure 2A-B). Furthermore, we analyzed the percentages of BrdU+ cells expressing DCX in the SGZ-GCL at 14 days following BrdU injections. We found that no difference in the percentages of BrdU+ cells expressing DCX between the three groups ( Figure 2C-D). While, the DCX+ cells decreased signi cantly in GWI rats compared to control rats, which indicated that the number of newly born immature neurons were diminished in the GWI rats, while this effect was blocked by minocycline treatment ( Figure 2E). Besides, we further analyzed the percentages of BrdU+ cells expressing NeuN in the SGZ-GCL at 28 days following BrdU injections. The data showed that no difference in the neuronal fate-choice decision by newly born cells between the three groups ( Figure  2F-G).

Minocycline treatment attenuated chronic systemic and brain in ammation in GWI rats
Previous studies revealed that exposure to Gulf war illness-related chemicals resulted in the immune system dysfunction 21 . Our results showed that the concentration of IL-17 was elevated both in serum and hippocampus of GWI rats and this increase was attenuated by minocycline treatment (Figure 3A, B). Moreover, the level of the anti-in ammatory marker of IL-10 in serum and hippocampus of GWI rats was signi cantly higher than the rats in the control group, but minocycline treatment increased the level of IL-10 signi cantly for GWI rats ( Figure 3C, D).
Furthermore, as previous studies reported that the TLR4/NF-κB signaling pathway is involved in the process of neuroin ammation 31 . Thus, to reveal the mechanism of minocycline treatment for regulation of neuroin ammation, we detected the expression of TLR4 and NF-κB p65 signaling pathway in hippocampus of rats after minocycline treatments by quantitative RT-PCR and western blotting (WB). Our results showed that the expression of TLR4 and NF-κB p65 were elevated signi cantly in GWI rats and these increases were attenuated by minocycline treatment (Figure 3E-H).

The effects of minocycline treatment on microbial diversity and the microbial community in GWI rats
As exposure of gulf war chemicals and the resultant chronic stress could alter the gut microbiome in the GWI animal model, we analyzed the composition of rat gut microbiota in stool samples by nextgeneration sequencing using V3-V4 hyper-variable 16S rRNA genomic region. Results showed that gulf war chemicals and the resultant chronic stress altered bacterial alpha diversity in the intestinal lumen signi cantly. Compared with the Control group, the GWI group had signi cantly increased commensal richness and diversity, as con rmed by the increased ACE index ( Figure 4A) and Shannon diversity ( Figure  4B). The ACE index and Shannon diversity was decreased in the GWI+Mino group compared with the GWI group ( Figure 4A-B). We also evaluated the dissimilarity between cecum bacterial communities among the groups by using principal coordinate analysis (PCoA) of the weighted UniFrac distance. The results showed that minocycline treatment reshaped the microbiota of rats with GWI ( Figure 4C). Moreover, we found that there were signi cant differences in the composition of gut bacteria both at the class and family levels between the groups ( Figure 4D-E). The class Bacilli, Erysipelotrichia and Deltaproteobacteria were underrepresented and Bacteroidia, Gammaproteobacteria, Spirochaetia and Mollicutes were overrepresented in GWI group compared with Control group, while minocycline treatment rescued these effects for rats with GWI ( Figure 4D). In addition, the abundance of the partial families that increased in GWI group, including Ruminococcaceae, Succinivibrionaceae, Lachnospiraceae, Prevotellaceae and Muribaculaceae, but Lactobacillaceae, Peptostreptococcaceae and Erysipelotrichaceae decreased. These changes in GWI rats also were rescued by minocycline treatment ( Figure 4E).

Discussion
In this study, we provided new evidence that minocycline treatment alleviated impaired cognitive and mood function in a rat model of GWI. Speci cally, we observed that these functional bene ts may are paralleled by changes in neuroin ammation, hippocampal neurogenesis and gut microbiome. To the best of our knowledge, our report is the rst to present comprehensive evidence that minocycline treatment is e cacious to alleviate GWI-related behaviors.
It has been shown that impairments in mood and cognitive function are the key brain abnormalities observed both in veterans with GWI and animal models 10,32 . The hippocampal pathology of GWI mainly include decreased neurogenesis, partial loss of principal neurons, mild in ammation and reduced hippocampal volume 10,33 . These changes in hippocampus showed an association with mood and cognitive dysfunction in a model of GWI. There are credible evidences to support the hypothesis that adult hippocampal neurogenesis in the hippocampus contributes to mood regulation. Especially, in some rodent models of depression and patients, reduced the size of hippocampus may be related to decreased neurogenesis and mature neuronal cell loss 34 . While the therapeutic action of antidepressants mainly depends on enhancing hippocampal neurogenesis and ablation of hippocampal neurogenesis impairs antidepressant e cacy 35,36 . Moreover, previous research has indicated that enhanced neurogenesis leads to better cognitive and mood function in a model of GWI 10 . Consistent with these ndings, we found minocycline treatment alleviated depressive-and anxiety-like behaviors and recognition memory in GWI rats. Meanwhile, substantial decreases in hippocampal neurogenesis were found in a model of GWI, while enhance hippocampal neurogenesis has been observed after minocycline treatment in rats with GWI. Remarkably, minocycline also showed the ability of enhance neurogenesis in others animal models, such as depression, schizophrenia and aging [37][38][39] . Thus, it is likely that the effects of minocycline on GWI-related behaviors may involve in enhance hippocampal neurogenesis.
Accumulating evidences suggest that chronic in ammation play a crucial role in regulating hippocampal neurogenesis 40 . Especially, neuroin ammation is one of the most essential factors in the progression of GWI. As previous reports, excessive neuroin ammation presented in veterans suffering from GWI and also observed in animal model of GWI 21,41 . Part of serum proteins associated with in ammation were signi cantly increased in veterans suffering from GWI, including interleukin 6, C-reactive protein, matrix metalloproteinase-9 (MMP-9) and matrix metalloproteinase-2 (MMP-2) 42,43 . In animal model of GWI, suppression of the in ammatory responses showed a neuroprotective effect, improved neurogenesis, and better cognitive and mood function 44 . Previous research has indicated that excessive neuroin ammation showed negative effect on neurogenesis in the hippocampus 18 . Interestingly, in the present study, we found minocycline treatment suppressed neuroin ammation in association with enhanced neurogenesis, better cognitive and mood function in animal models of GWI, indicating a linkage between these processes. Moreover, TLR4/NF-κB pathway in the most important regulators of in ammatory cascades in neuroin ammation. It has been reported that activation of the TLR4/NF-κB pathway leads to acute activation of microglia, which results in the release of pro-in ammatory cytokines. In our study, we have observed that minocycline treatment can reverse increased levels of TLR4 and NF-κB in hippocampus of rats with GWI. Consistently, previous studies also have revealed the anti-in ammatory effect of minocycline in other models of neurodegenerative and neuroin ammatory diseases, including Alzheimer's disease, depression, intracerebral hemorrhage and etc. 45 Thus, our results suggest that minocycline show an anti-in ammatory effect by interrupting of the TLR4/NF-κB signaling pathway.
Studies have shown that stress can change the composition of gut microbiota, and disruptions of the gut microbiota contribute to the changes of systemic in ammation, behaviors, and cognitive function 46,47  gut and systemic endotoxemia-induced TLR4 activation contributes to GWI chemical-induced neuroin ammation and gastrointestinal disturbances. Evidence found in our study showed that a direct relationship of gulf war chemical exposure and altered gut microbiota diversity with changes in the Phylum and family levels.

Conclusion
In summary, our study provided the rst evidence that minocycline treatment alleviated impaired cognitive and mood function via reversing gut dysbiosis, the dysregulation of the neuroin ammation and restoring hippocampal neurogenesis in a rat model of GWI. Our study suggested that close attention should be paid to the link between GWI and dysbiosis. Our ndings provide evidence that minocycline can be a novel strategy that are e cacious for repairing gut dysbiosis, suppressing in ammation and enhancing neurogenesis may be helpful for improving mood and cognitive function in veterans with GWI.