Prasad S Dalvi

Jennifer A Chalmers

Vicky Luo

D-Yd Han

Leigh Wellhauser

Ying Liu

Dean Q Tran

Julien Castel

Serge Luquet

Michael B Wheeler

PhD Denise D Belsham
email: d.belsham@utoronto.ca

Dao-Yu D Han

High-fat induces acute and chronic inflammation in the hypothalamus: Effect of HFD, palmitate and TNF-a on appetite-regulating NPY neurons

Keywords: hypothalamus, neuroinflammation, palmitate, TNF-alpha, neuropeptide Y, cytokines, transcription, signal transduction

High fat induces acute and chronic inflammation in the

hypothalamus: effect of high-fat diet, palmitate and TNF-alpha on appetite-regulating NPY neurons

Introduction

Obesity has emerged as a major health problem not only in the industrialized countries, but also in the developing nations. Sedentary lifestyles, reduced physical activity, accompanied by increased consumption of dietary fats, have contributed to the worldwide obesity pandemic [START_REF] Flier | Obesity wars: molecular progress confronts an expanding epidemic[END_REF] .

Food intake and energy expenditure are tightly regulated by the hypothalamus. Hypothalamic appetite-regulating neurons, such as neuropeptide Y (NPY), agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) neurons, are responsible for sensing information on nutritional status by integrating central and peripheral signals [START_REF] Niswender | Intracellular signalling. Key enzyme in leptin-induced anorexia[END_REF] . Recent studies have shown that consumption of dietary fats induces hypothalamic resistance to insulin and leptin, which contributes to hyperphagia, obesity, and type 2 diabetes (T2DM) [START_REF] Milanski | Saturated fatty acids produce an inflammatory response predominantly through the activation of TLR4 signaling in hypothalamus: implications for the pathogenesis of obesity[END_REF][START_REF] Souza | Consumption of a fat-rich diet activates a proinflammatory response and induces insulin resistance in the hypothalamus[END_REF] . In rodent genetic and diet-induced obesity models, the functional resistance to insulin and leptin in the hypothalamus develops partly through activation of inflammation in the mediobasal hypothalamus [START_REF] Milanski | Saturated fatty acids produce an inflammatory response predominantly through the activation of TLR4 signaling in hypothalamus: implications for the pathogenesis of obesity[END_REF][START_REF] Souza | Consumption of a fat-rich diet activates a proinflammatory response and induces insulin resistance in the hypothalamus[END_REF][START_REF] Howard | Enhanced leptin sensitivity and attenuation of diet-induced obesity in mice with haploinsufficiency of Socs3[END_REF] . Additionally, research using rodent strains sensitive to diet-induced obesity has demonstrated an altered hypothalamic NPY/AgRP content or expression [START_REF] Lemus | A stereological analysis of NPY, POMC, Orexin, GFAP astrocyte, and Iba1 microglia cell number and volume in diet-induced obese male mice[END_REF][START_REF] Wang | Effects of dietary fat types on body fatness, leptin, and ARC leptin receptor, NPY, and AgRP mRNA expression[END_REF] , however, the exact role of inflammation in these neurons in obesity is poorly understood. Another study using postmortem human hypothalamii indicated that the expression of AgRP and NPY is correlated well with body weight changes or body mass index [START_REF] Alkemade | AgRP and NPY expression in the human hypothalamic infundibular nucleus correlate with body mass index, whereas changes in alphaMSH are related to type 2 diabetes[END_REF] , yet the contribution of neuroinflammation in regulation of these neuropeptides still remains unstudied.

In recent years, a number of studies have implicated several signal transduction pathways involved in the induction of inflammation, such as suppressor of cytokine signaling-3 (SOCS3) [START_REF] Howard | Enhanced leptin sensitivity and attenuation of diet-induced obesity in mice with haploinsufficiency of Socs3[END_REF] , c-Jun N-terminal kinase (JNK) and I kappa B kinase (IKK) [START_REF] Souza | Consumption of a fat-rich diet activates a proinflammatory response and induces insulin resistance in the hypothalamus[END_REF] . Further, several studies have provided considerable evidence that the inflammatory response to dietary fat is mediated by tolllike receptor (TLR) signaling, activation of NF-κB, and production of inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and TNF-α [START_REF] Akira | Toll-like receptor signalling[END_REF][START_REF] Hayden | Shared principles in NF-kappaB signaling[END_REF] . Among these inflammatory markers, a role for TNF-α in the pathogenesis of T2DM and obesity has been demonstrated [START_REF] Miyazaki | Tumor necrosis factor alpha and insulin resistance in obese type 2 diabetic patients[END_REF] . In diet-induced obese rats, the expression of TNF-α and other proinflammatory cytokines is remarkably increased in the hypothalamus [START_REF] Souza | Consumption of a fat-rich diet activates a proinflammatory response and induces insulin resistance in the hypothalamus[END_REF][START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF] , and TNF-α has been found to modulate hypothalamic neuropeptides involved in appetite regulation [START_REF] Amaral | Tumor necrosis factor-alpha activates signal transduction in hypothalamus and modulates the expression of pro-inflammatory proteins and orexigenic/anorexigenic neurotransmitters[END_REF] . However, the temporal activation of TNF-α and other proinflammatory markers by acute and chronic high-fat diet (HFD) in the hypothalamic mediobasal areas that are critical for energy homeostasis remains uncharacterized.

© 2016 Macmillan Publishers Limited. All rights reserved.

In the present study, we confirmed acute upregulation of TNF-α expression and astrocytosis in the arcuate nucleus (ARC) within 24 h of Intralipid infusion, which corroborates a previous study using HFD [START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF] . Within the first 8 weeks of chronic HFD feeding, the TNF-α expression was reduced; however, the expression of heat shock protein 70 (HSP70) that protects from oxidative stress, and also the expression of ciliary neurotrophic factor (CNTF) that reduces neuronal injury during inflammation, was increased, suggesting an effective neuroprotective response in the ARC of the hypothalamus. The TNF-α expression increases following long-term HFD feeding for 20 weeks indicating that TNF-α-induced inflammation may have a timedependent neurotrophic or neurotoxic role. Using well-established mouse hypothalamic cell lines, we report evidence of increased inflammatory and ER stress markers with acute treatment of the saturated fatty acid palmitate and TNF-α. We also report that appetite-stimulating NPY is significantly upregulated by palmitate and TNF-α in the neuronal cells. These in vivo and in vitro findings collectively suggest that acute and chronic HFD exposure regulates hypothalamic inflammation by temporal activation of TNF-α that may further modulate appetite by positively regulating NPY, suggesting a dysregulation of energy homeostasis feedback in a high fat environment.

Methods

Animals

Animal experiments were performed with approval of the Animal Care Committee of the University Paris Diderot-Paris 7 and the University of Toronto. Male C57BL/6 mice (Janvier, Le Genest St Isle, France) were housed individually. Male CD-1 mice (Charles River, Canada) were maintained on an ad libitum standard rodent chow diet. C57BL/6 mice were used for acute Intralipid infusion, and CD-1 mice were fed with a diet containing 60% kcal fat (HFD, 5.49 kcal/gm, F3282; Bio-Serv, NJ, USA) for periods ranging from 8-20 weeks. 10 to 12 week old animals were used at the commencement of the experiments. Once the mice were switched to the HFD feeding, body weight was monitored weekly.

Intralipid Infusion

© 2016 Macmillan Publishers Limited. All rights reserved.

Catheter implantation and lipid perfusion were carried out as previously described [START_REF] Cansell | Dietary triglycerides act on mesolimbic structures to regulate the rewarding and motivational aspects of feeding[END_REF] .

Infusions started after a 7-10 days recovery period. Intralipid® was obtained from Sigma-Aldrich (St. Louis, MO). Mice were infused with saline solution for 3-4 days for habituation to the infusion device. Mice were then divided into 2 groups: one received saline and the other a triglyceride emulsion Intralipid at a rate of 0.1-0.3 µl/min for 6 h. The pump settings allowed a constant flow rate of ~0.1 µl/min. The animals were sacrificed 24 h following infusion.

Glucose tolerance test:

Glucose tolerance was assessed by intraperitoneal (i.p.) glucose tolerance test (IPGTT) after 7 weeks of treatment. Mice were fasted for 6 h, and the tests were carried out at 2:00 pm.

Glucose (1.5 mg/g body weight) was administered by i.p. injection. Blood samples were drawn from the tail vein at 0, 10, 30, 60, 90, and 120 min after glucose administration. Blood glucose levels were measured using a Glucometer (Bayer).

Immunofluorescence

Mice were anesthetized and perfused transcardially with ice-cold phosphate-buffered saline followed by freshly prepared 4% paraformaldehyde solution. The brains were removed, post-fixed in 4% paraformaldehyde, serially cryoprotected in sucrose, snap-frozen in and stored at -80°C. Frozen brains were sliced in 20 μm sections (Leica CM1510S), as previously described [START_REF] Dalvi | Glucagon-like peptide-1 receptor agonist, exendin-4, regulates feeding-associated neuropeptides in hypothalamic neurons in vivo and in vitro[END_REF] . Sections blocked in 5% normal donkey or rabbit serum (Vector Laboratories, Burlington, ON) were incubated overnight at 4ºC with specific primary antibodies: rabbit anti-AgRP (1:200; catalog no. #H003-57, Phoenix Pharmaceuticals, Burlingame, CA), rabbit anti-NPY (1:200; catalog no. #H049-03, Phoenix Pharmaceuticals), chicken anti-NSE (1:100; catalog no. #Ab 39369, Abcam, Cambridge, MA), chicken anti-GFAP (1:500; catalog no. #Ab 4674, Abcam), goat anti-TNF-α (1:50; catalog no. #SC1350, Santa Cruz, Inc., Santa Cruz, CA), rabbit anti-HSP70 (1:100; catalog no. Ab31010, Abcam), and rabbit anti-CNTF (1:1000; catalog no. Ab46172, Abcam). Immunofluorescence was performed with a combination of Alexa Flour 555or Alexa Flour 488-labeled anti-rabbit, anti-goat, or anti-chicken secondary antibodies (1:500; Life Technologies Inc.) and nuclear stain TOPRO (1:1000; catalog no. T3605(Life Technologies Inc.) was used to identify cell nuclei.

© 2016 Macmillan Publishers Limited. All rights reserved.

Images from immunostained sections were captured on a Zeiss LSM 510 confocal upright microscope outfitted with a color digital camera and AxioVision 3.0 imaging software. For the quantification of cells, every second section throughout the ARC was taken to visualize the distribution of immuofluorescence (total 4-5 sections/mouse). For each of the sections, bilateral images were captured in a single plane of focus at x40 magnification and a 100 µm 2 box was placed in the center of the selected hypothalamic ARC region. The intensity of immunofluorescence was measured in a blinded manner using ImageJ software. The intensity was determined as the signal to background intensity ratio. The results were expressed as relative intensity of GFAP, TNF-α, CNTF, HSP70 or NSE in hypothalamic ARC AgRP-or NPYexpressing neurons for each group of mice.

Cell Models

The clonal, embryonic mouse hypothalamic mHypoE-44 and mHypoE-46 cell lines, and the adult mouse hypothalamic mHypoA-NPY/GFP cell line, were generated and culture conditions described previously by our laboratory [START_REF] Belsham | Generation of a phenotypic array of hypothalamic neuronal cell models to study complex neuroendocrine disorders[END_REF][START_REF] Belsham | Ciliary neurotrophic factor recruitment of glucagon-like peptide-1 mediates neurogenesis, allowing immortalization of adult murine hypothalamic neurons[END_REF][START_REF] Dhillon | Cellular leptin resistance impairs the leptin-mediated suppression of neuropeptide Y secretion in hypothalamic neurons[END_REF] . Cell lines were in 60 mm dishes (Sarstedt, Montreal, QC)and allowed to reach 70-80% confluence. TNF-α, sodium palmitate and the IKKβ inhibitor, PS1145 were purchased from Sigma. Sterile hypure water was used as the vehicle for TNF-α and sodium palmitate. Sodium palmitate was solubilized in sterile hypure water by heating the mixture to 65°C followed by gentle vortexing, and immediately added to culture medium pre-heated to 37°C containing 5% FBS to act as a carrier. The inhibitor PS1145 was prepared in dimethyl sulfoxide (DMSO), and the concentration of DMSO was ~0.01% for treatments. Cells were pre-treated with PS1145 1 hour prior to administration of TNF-α or sodium palmitate.

Quantitative RT-PCR

RNA was isolated from cell using the purelink RNA mini columns and on column DNAse (Ambion). A total of 1 ug of RNA was then used to synthesize cDNA with the high capacity cDNA archive kit (Applied Biosystems). Quantitative RT-PCR was conducted using gene specific primers (Supplemental Table 1) and platinum SYBR green qPCR supermix-UDG w/ROX according to the manufacturer's instructions. cDNA samples (25 ng) and dose curve (Range of 50 ng to 1.56 ng) were loaded in triplicate for each gene. Samples were run on a 384-© 2016 Macmillan Publishers Limited. All rights reserved.

well block ABI 7900HT fast real-time PCR system at 50°C for 2 min, 95°C for 2 min, and 40 repeats of 95°C for 15 sec to 60°C for 1 min, followed by melting curve analysis at 95°C for 15 sec to 60°C for 15 sec then 95°C for 15 sec. and analyzed using SDS 2.4 software (Applied Biosystems).

Statistical analysis

Data are presented as the mean ± SEM. Data were analyzed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA). Two groups were compared using two-tailed Student's t tests. For more than two groups, statistical analysis was performed using one-way or two-way ANOVA, and statistical significance was determined by post hoc analysis using Bonferroni test, Holm-Sidak or Student's t-test with P < 0.05.

Results

Temporal expression of inflammatory markers

To test whether acute treatment with fatty acids induces hypothalamic inflammation, we used Intralipid, an emulsion of fatty acids containing 7-14% palmitic acid together with a mixture of linoleic and oleic acids. We injected Intralipid via carotid artery cannulation for its direct delivery into the brain and these lipids readily diffuse across the blood-brain barrier to access hypothalamic neurons [START_REF] Hamilton | Fatty acid transport: the diffusion mechanism in model and biological membranes[END_REF] . We found that TNF-α immunoreactivity was significantly increased within 24 h post fatty acid exposure in the hypothalamus (Figures 1A-C). We observed no change in the immunoreactivity of either CNTF or HSP70 (Figure 1C). We found that the number of GFAP-positive astrocytes was singificantly upregulated (Figure 1D).

To further understand the effect of prolonged exposure to saturated fats on hypothalamic inflammation and putative neuroprotective responses, mice were fed a HFD for 8 weeks. HFDfed mice exhibited significantly increased body weight gain over time (Figure 1E), however we found that the cumulative food intake during the 8th week of feeding was significantly decreased in HFD-fed mice (Figure 1F). To test the efficacy of the HFD feeding on induction of glucose intolerance, we performed IPGTTs. When glucose was administered via an i.p. injection, HFDfed mice demonstrated significantly increased glucose excursion and area under the glucose © 2016 Macmillan Publishers Limited. All rights reserved.

curve compared with chow diet-fed mice (Figures 1G andH), suggesting dysregulation of glucose homeostasis in the HFD-fed mice. Further, we found that the immunofluorescence of TNF-α was significantly diminished at this stage (Figures 1I-K). On the other hand, we found a significant increase in the expression of HSP70 (Figure 2A-C) and CNTF (Figure 2D-F), the prominent components of the neuroprotective response to neuronal injury. We also found that the number of GFAP-positive astrocytes remained singificantly upregulated (Figure 2G). In a separate cohort of mice fed with HFD for 8 weeks, gene expression analysis revealed significantly elevated hypothalamic levels of IĸBα, without any change in the levels of IL-6 and NPY, however, the levels of AgRP and NF-κB, an activator of TNF-α, were significantly suppressed (Figure 2H).

In mice fed a HFD for 20 weeks, the expression of TNF-α was found to be significantly elevated in the ARC NPY/AgRP neurons (Figures 3A-C). To further demonstrate that the hypothalamic inflammation was clearly evident in the ARC neurons, and not only limited to the glia, we used immunofluorescence for neuron-specific enolase (NSE), marker of mature neurons. We detected that TNF-α and NSE were clearly co-localized in the ARC (Figure 3D).

Astrocytosis marker analysis

During neuronal injury, astrocytes can be activated and mobilized to the site of the injury where they can further proliferate to give rise to reactive astrocytosis [START_REF] Sofroniew | Molecular dissection of reactive astrogliosis and glial scar formation[END_REF][START_REF] Ransohoff | Microglial physiology: unique stimuli, specialized responses[END_REF] . Using immunohistochemistry to detectimmunoreactivity of GFAP, which is regarded as a sensitive and reliable marker expressed by most reactive astrocytes that are responding to CNS injuries [START_REF] Sofroniew | Astrocytes: biology and pathology[END_REF] , we found that there was a significant upregulation of GFAP expression by 17% in the hypothalamic ARC by 24 h following Intralipid infusion in mice (Figures 1A-D). This result suggests an acute effect of fatty acids in promoting astrocyte accumulation in this brain region. Interestingly, the level of hypothalamic astrocytosis was found to be elevated by 135%-290% following extended HFD feeding at 8 weeks compared with that in chow-fed controls (Figures 1I-K and2A-G).

Temporal palmitate-induced gene expression

Intralipid contains 7-14% palmitate and the HFD used to chronically feed the mice contained 14.1% saturated fat. Therefore, to further understand the role of saturated fatty acid-© 2016 Macmillan Publishers Limited. All rights reserved.

induced acute inflammation in appetite-regulating NPY/AgRP neurons, we used the saturated fatty acid palmitate to treat immortalized mouse hypothalamic cell lines, expressing NPY and AgRP, for the in vitro experimentation [START_REF] Belsham | Generation of a phenotypic array of hypothalamic neuronal cell models to study complex neuroendocrine disorders[END_REF] . Expression of pro-inflammatory markers, such as IBα, NF-κB p50 subunit, IL-6, TNF-α; NF-κB p65 subunit RelA; pro-apoptotic proteins, such as CHOP, p53, FADD, glucose-regulated protein-78; components of the neuroprotective response to neuron injury, such as HSP70, HSP72; and appetite-regulating genes, such as AgRP and NPY, were assessed by qRT-PCR. Palmitate treatment (4 h) in the hypothalamic mHypoE-44 and -46 neuronal cells induced a mixed inflammatory response, while robustly increasing mRNA levels of CHOP, a marker of endoplasmic reticulum (ER) stress implicated in apoptosis, and significantly upregulated NPY mRNA levels without any change in AgRP mRNA (Figures 4A andB). Of interest, we did not observe any change in the level of TNF-α mRNA in either cell line; however, IBα and NF-κB mRNA levels decreased, whereas proinflammatory marker IL-6 mRNA levels changed in a cell-specific manner.

Prolonged exposure to palmitate for up to 33 h demonstrated that NPY (Figures 5A andB) and CHOP (Figures 5C andD) mRNA levels in both mHypoE-44 and -46 cell lines remain elevated, whereas AgRP mRNA levels again did not change (data not shown). By inhibiting the IKKβ/NF-κB cascade through pre-treatment with the IKKβ inhibitor PS1145 (20 M), we demonstrated that the palmitate-mediated increase in NPY mRNA levels was abolished, indicating palmitate acts through the IKKβ/NF-κB pathway to regulate NPY expression (Figures 6A andB). On the other hand, CHOP mRNA levels were unchanged, suggesting an IKKβ/NF-κB -independent mechanism forits upregulation (Figures 6C andD).

Temporal TNF-α-induced gene expression

Based on the acutely increased expression of TNF-α in vivo, we further investigated the potential role of TNF-α on acute inflammation in the NPY/AgRP neuronal cell models. Besides the mHypoE-46 cell line [START_REF] Belsham | Generation of a phenotypic array of hypothalamic neuronal cell models to study complex neuroendocrine disorders[END_REF] , we used another well-characterized adult-derived mouse NPY/AgRP cell line, mHypoA-NPY/GFP [START_REF] Dhillon | Cellular leptin resistance impairs the leptin-mediated suppression of neuropeptide Y secretion in hypothalamic neurons[END_REF] . We found that when treated with TNF-α, both the hypothalamic cell lines demonstrated a robust inflammatory response indicated by an upregulation of IBα, NF-κB, IL-6, and RelA mRNA levels within 6 h following TNF-α treatment (Figures 4C andD). This upregulation was found to be independent of the IKKβ/NF-κB cascade given that PS1145 © 2016 Macmillan Publishers Limited. All rights reserved.

pretreatment did not abolish the effect of TNF-α (Figures 6H andJ). Treatment with TNF-α in the mHypoE-46 cells, but not in the mHypoA-NPY/GFP cells, upregulated NPY mRNA levels within 6 h, which was reversed by pretreatment with PS1145, indicating involvement of the inflammatory IKKβ/NF-κB pathway, similar to that found with palmitate (Figures 6E andF).

Discussion

Activation of an inflammatory response in the hypothalamus is well-documented in rodent models of diet-induced obesity [START_REF] Milanski | Saturated fatty acids produce an inflammatory response predominantly through the activation of TLR4 signaling in hypothalamus: implications for the pathogenesis of obesity[END_REF][START_REF] Souza | Consumption of a fat-rich diet activates a proinflammatory response and induces insulin resistance in the hypothalamus[END_REF][START_REF] Zhang | Hypothalamic IKKbeta/NF-kappaB and ER stress link overnutrition to energy imbalance and obesity[END_REF] , however, the underlying mechanisms remain largely unstudied. Further, the temporal regulation of TNF-α and other markers of neuron injury or protection are not yet described. We report that the expression of TNF-α, HSP70, and CNTF in the hypothalamus is temporally regulated by Intralipid infusion or HFD feeding in mice. A rapid increase in TNF-α expression in the ARC following an acute infusion of Intralipid suggests a primary response that most likely limits or reverses the injury caused by high fatty acid exposure in favour of neuroprotection, despite being a proinflammatory cytokine [START_REF] Cheng | Tumor necrosis factors protect neurons against metabolic-excitotoxic insults and promote maintenance of calcium homeostasis[END_REF][START_REF] Marchetti | Tumor necrosis factor (TNF)mediated neuroprotection against glutamate-induced excitotoxicity is enhanced by Nmethyl-D-aspartate receptor activation. Essential role of a TNF receptor 2-mediated phosphatidylinositol 3-kinase-dependent NF-kappa B pathway[END_REF] . The neuroprotective role of TNF-α is also demonstrated in TNF-α receptor-deficient mice that develop increased neuronal lesions in response to ischemic injury [START_REF] Bruce | Altered neuronal and microglial responses to excitotoxic and ischemic brain injury in mice lacking TNF receptors[END_REF] . The suppression of TNF-α expression and increase in HSP70 and CNTF that were observed with extended exposure to HFD feeding at 8 weeks suggest possible neuroprotective or even neuroregenerative responses that can be triggered to further prevent or limit injury and promote repair mechanisms in the hypothalamus [START_REF] Belsham | Ciliary neurotrophic factor recruitment of glucagon-like peptide-1 mediates neurogenesis, allowing immortalization of adult murine hypothalamic neurons[END_REF][START_REF] Turturici | Hsp70 and its molecular role in nervous system diseases[END_REF][START_REF] Kokoeva | Neurogenesis in the hypothalamus of adult mice: potential role in energy balance[END_REF] . Eventually, chronic HFD feeding at 20 weeks causes an increase in TNF-α expression in the NPY/AgRP neurons, suggesting exhaustion of hypothalamic protective mechanisms and development of chronic inflammation, favouring appetite and neuropeptide dysregulation inclined towards energy intake.

Acute Intralipid infusion induces TNF-α expression in the hypothalamic neurons within 24 h. Although some previous studies have evaluated the abilities of fatty acids to induce cytokine expression in the hypothalamus [START_REF] Milanski | Saturated fatty acids produce an inflammatory response predominantly through the activation of TLR4 signaling in hypothalamus: implications for the pathogenesis of obesity[END_REF] , this is the first description of TNF-α induction in the mouse hypothalamus within a short period of time. Our finding is consistent with the previously observed HFD-induced acute increase in TNF-α mRNA expression that was accompanied with reactive gliosis in the hypothalamus of rats after one day of HFD feeding [START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF] . The same study found that the acutely increased TNF-α mRNA expression eventually subsided only to rise again © 2016 Macmillan Publishers Limited. All rights reserved. after 4 weeks of HFD feeding [START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF] . The rapid onset of inflammation in the mediobasal hypothalamus manifests neuron injury that may recruit neuroprotective measures to prevent further damage of the ARC neurons. This initial neuroprotective response represents an important defence mechanism to protect neurons involved in appetite regulation and energy homeostasis. As such, it has already been demonstrated that chronic HFD feeding significantly reduces the number of hypothalamic POMC neurons that play a major role in protecting against excess weight gain 12 , and their loss is sufficient to cause obesity in mice [START_REF] Gropp | Agouti-related peptide-expressing neurons are mandatory for feeding[END_REF] .

Although, the exact role of Intralipid-induced acute TNF-α in the hypothalamus remains to be investigated, Amaral et al. evaluated the capacity of TNF-α to modulate hypothalamic neuropeptides involved in appetite regulation and energy homeostasis [START_REF] Amaral | Tumor necrosis factor-alpha activates signal transduction in hypothalamus and modulates the expression of pro-inflammatory proteins and orexigenic/anorexigenic neurotransmitters[END_REF] , in which they found that direct TNF-α injection in the hypothalamus acutely upregulates both orexigenic NPY and anorexigenic POMC expression within 2 h post-injection [START_REF] Amaral | Tumor necrosis factor-alpha activates signal transduction in hypothalamus and modulates the expression of pro-inflammatory proteins and orexigenic/anorexigenic neurotransmitters[END_REF] . Acute TNF-α infusion has also been shown to cause hypothalamic insulin and leptin resistance within 4 h post-infusion [START_REF] Picardi | Modulation of hypothalamic PTP1B in the TNF-alpha-induced insulin and leptin resistance[END_REF] . We did not investigate whether Intralipid or the Intralipid-induced increase in the TNF-α expression causes neuronal insulin resistance in these mice; however, studies have demonstrated that a short exposure to fatty acids results in a significant reduction in insulin signaling in the CNS [START_REF] Holland | Lipidinduced insulin resistance mediated by the proinflammatory receptor TLR4 requires saturated fatty acid-induced ceramide biosynthesis in mice[END_REF][START_REF] Benoit | Palmitic acid mediates hypothalamic insulin resistance by altering PKC-theta subcellular localization in rodents[END_REF] .

Overall, based on the past and present evidence we predict a possible path towards the development of insulin resistance via TNF-α induction following acute Intralipid treatment in mice, and therefore, further studies are needed to understand the mechanistic role of TNF-α in this process.

The HFD used contained as much as 6-fold more saturated fat than the control diet. This is comparable to the amount of saturated fat present in most Western diets [START_REF] Dougherty | Nutrient content of the diet when the fat is reduced[END_REF] . Chronic feeding of rats with a similar diet has been shown to cause high levels of TNF-α, IL-1β, and IL-6 in the hypothalamus, predominantly in the median eminence and ARC [START_REF] Milanski | Saturated fatty acids produce an inflammatory response predominantly through the activation of TLR4 signaling in hypothalamus: implications for the pathogenesis of obesity[END_REF] . It was found that elevated levels of pro-inflammatory cytokines, such as TNF-α, induce cell apoptosis that can be prevented by over-expression of HSP70 [START_REF] Gao | Heat shock protein 70 together with its co-chaperone CHIP inhibits TNF-alpha induced apoptosis by promoting proteasomal degradation of apoptosis signal-regulating kinase1[END_REF] . In the brain, the heat shock proteins serve a neuroprotective role [START_REF] Sharp | Heat-shock protein protection[END_REF] . HSP70 is well-studied and is involved in the repair mechanism after environmental stressors, such as heat, ischemia, ultraviolet irradiation, and oxidative stress [START_REF] Sharp | Heat-shock protein protection[END_REF] . In the present study, the increase in HSP70 that was observed following extended exposure to HFD feeding supports © 2016 Macmillan Publishers Limited. All rights reserved.

itsneuroprotective role in the hypothalamus. Another study found that the mRNA expression of hypothalamic HSP72, a member of the HSP70 family, was increased within 3 days of HFD exposure in rats and was detected in the POMC neurons that are critical for energy balance regulation [START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF] . The induction of heat shock proteins following HFD warrants further study on their potential therapeutic neuroprotective roles.

Studies have documented the ability of glial-derived CNTF to promote cell survival and differentiation in a number of neuronal and glial cell types [START_REF] Sendtner | Ciliary neurotrophic factor[END_REF][START_REF] Sleeman | The ciliary neurotrophic factor and its receptor, CNTFR alpha[END_REF] . Here we show that CNTF expression does not change in the hypothalamic ARC following acute Intralipid infusion, but in 8 week HFD feeding conditions it significantly increases in the ARC of mice. Our immunofluorescence data indicate that an increase in CNTF expression was paralleled by an increase in hypothalamic astrocyte expression, suggesting that chronic HFD feeding in mice is associated with increased CNTF expression via astrocytosis in the hypothalamus. This finding supports the notion that CNTF is a novel, glial-derived modulator of hypothalamic neuropeptidergic neurons in chronic HFD-fed mice [START_REF] Severi | Opposite effects of a high-fat diet and calorie restriction on ciliary neurotrophic factor signaling in the mouse hypothalamus[END_REF] . Studies have demonstrated a neuroprotective effect of CNTF via stimulation of gene expression, cell survival or differentiation [START_REF] Sleeman | The ciliary neurotrophic factor and its receptor, CNTFR alpha[END_REF] . Recently, we and others have shown that CNTF induces neurogenesis in the adult hypothalamus and may contribute to the control of energy homeostasis [START_REF] Belsham | Ciliary neurotrophic factor recruitment of glucagon-like peptide-1 mediates neurogenesis, allowing immortalization of adult murine hypothalamic neurons[END_REF][START_REF] Kokoeva | Neurogenesis in the hypothalamus of adult mice: potential role in energy balance[END_REF] . Other studies have also clearly demonstrated that administration of CNTF or its recombinant analog Axokine reduces food intake [START_REF] Gloaguen | Ciliary neurotrophic factor corrects obesity and diabetes associated with leptin deficiency and resistance[END_REF][START_REF] Lambert | Ciliary neurotrophic factor activates leptin-like pathways and reduces body fat, without cachexia or rebound weight gain, even in leptin-resistant obesity[END_REF][START_REF] Bluher | Ciliary neurotrophic factorAx15 alters energy homeostasis, decreases body weight, and improves metabolic control in diet-induced obese and UCP1-DTA mice[END_REF] . Thus, the increased expression suggests that CNTF could potentially enhance neurogenesis in the hypothalamus, as a part of reactive repair mechanism to counter the detrimental effects of HFD exposure on neurons.

We found that long-term HFD feeding in mice induced increased levels of TNF-α expression in NPY/AgRP neurons. Studies on enteric neurons have suggested that during inflammation TNF-α upregulation induces NPY, and NPY-TNF-α crosstalk aggravates inflammatory signaling [START_REF] Chandrasekharan | Tumor necrosis factor (TNF)-neuropeptide Y (NPY) crosstalk regulates inflammation, epithelial barrier functions and colonic motility[END_REF] . In support of this previous finding, our study demonstrates a high level of crosstalk between NPY-TNF-α in the NPY/AgRP neurons in favour of excessive energy accumulation in HFD-fed mice. However, we found that in chronically HFD-fed mice the cumulative food intake decreased over time suggesting a compensatory response to surplus energy balance, but the neuronal circuits involved and the exact role and contribution of the © 2016 Macmillan Publishers Limited. All rights reserved.

TNF-α-mediated inflammation in the arcuate NPY/AgRP neurons in the chronic positive energy balance despite reduced food intake warrants further investigation. To further enhance our understanding of the acute effect of palmitate or TNF-α on the markers of inflammation or neuropeptide expression, we selected three different cell lines that endogenously express NPY/AgRP [START_REF] Belsham | Generation of a phenotypic array of hypothalamic neuronal cell models to study complex neuroendocrine disorders[END_REF][START_REF] Dhillon | Cellular leptin resistance impairs the leptin-mediated suppression of neuropeptide Y secretion in hypothalamic neurons[END_REF] . Accordingly, we found that both palmitate and TNF-α induce inflammatory markers in these neuronal cells with simultaneous upregulation of NPY mRNA levels. Palmitate also induced a robust increase in the CHOP mRNA, a marker of endoplasmic reticulum (ER) stress implicated in apoptosis. Indeed, saturated fatty acids induce ER stress via activation of TLR signaling in the hypothalamus [START_REF] Milanski | Saturated fatty acids produce an inflammatory response predominantly through the activation of TLR4 signaling in hypothalamus: implications for the pathogenesis of obesity[END_REF] . Furthermore, in diet-induced obese mice, CHOP and other markers of ER stress were linked in the hypothalamus to an induction of central leptin and insulin resistance [START_REF] Won | Central administration of an endoplasmic reticulum stress inducer inhibits the anorexigenic effects of leptin and insulin[END_REF] . Previously, using the mHypoE-44 cell line, it was found that palmitate treatment induced ER stress through a c-Jun N-terminal kinase (JNK)-dependent pathway and activation of the ER stress markers eIF2 alpha and X-box binding protein-1 to induce insulin resistance [START_REF] Mayer | Palmitate attenuates insulin signaling and induces endoplasmic reticulum stress and apoptosis in hypothalamic neurons: rescue of resistance and apoptosis through adenosine 5' monophosphate-activated protein kinase activation[END_REF] . However, whether CHOP activation in these neuronal cells induces leptin or insulin resistance or apoptosis remains to be studied. TLR4 acts as a predominant molecular target for saturated fatty acids in the hypothalamus, triggering the intracellular signaling network that induces an inflammatory response via upregulation of TNF-α, and determines the resistance to anorexigenic or susceptibility to orexigenic signals [START_REF] Milanski | Saturated fatty acids produce an inflammatory response predominantly through the activation of TLR4 signaling in hypothalamus: implications for the pathogenesis of obesity[END_REF] . The findings from this study indicate that acute treatment of TNF-α induces robust inflammatory response by upregulating IBα, NF-κB and IL-6 mRNA levels in the hypothalamic neuronal models. These components are a part of the IKKβ/NF-κB cascade activated by TLR4 [START_REF] Akira | Toll-like receptor signalling[END_REF][START_REF] Hayden | Shared principles in NF-kappaB signaling[END_REF] . With the IKKβ inhibitor PS1145, we demonstrated that the palmitate-mediated increase in NPY mRNA levels was abolished, indicating palmitate and TNFα act through the IKKβ/NF-κB pathway to regulate NPY expression. However, the exact downstream mechanisms responsible for the change in NPY mRNA expression remain to be determined. Further studies are also warranted in other neuronal cell types to understand the role of activated TNF-α together with robust astrocytosis in the neurodegeneration of anorexigenic neurons, such as POMC during chronic HFD feeding conditions [START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF][START_REF] Moraes | High-fat diet induces apoptosis of hypothalamic neurons[END_REF] . Clearly, there will likely be a coordinated activation of pathways in each neuronal cell type to achieve the final detrimental result of chronic obesity.

© 2016 Macmillan Publishers Limited. All rights reserved.

In conclusion, we report that, in mice, acute exposure to fatty acids induces expression of inflammatory cytokine TNF-α in the mediobasal hypothalamus that is accompanied with reactive astrocytosis. The longer-term HFD exposure at 8 weeks does not induce TNF-α expression, but gives rise to induction of markers of neuroprotection and neuroregeneration. Although the defence mechanisms appear to limit the neuronal injury initially, recovery appears to be transient as chronic inflammation rebounds with extended HFD feeding. Reactive astrocytosis remains persistent with the HFD feeding model most probably due to neuronal injury, damage, or loss in the hypothalamus critical for energy homeostasis. The in vitro data suggests that TNF-α may play an important role in palmitate-and TNF-α-induced acute or longer-term stimulation of NPY via activation of the inflammatory IKKβ/NF-κB cascade. These findings suggest a chronic positive energy balance mechanism after prolonged exposure to HFD or saturated fatty acids, such as palmitate, mediated through the increased expression of the appetite-stimulating neuropeptide, NPY, and this could ultimately lead to the difficulties experienced with weight regulation and management. following Intralipid infusion in C57BL/6 mice, whereas the number of GFAP-positive cells was significantly upregulated (D). (E, F) Analysis revealed that 8 weeks of HFD feeding had significant effect on body weight gain (E), while the cumulative food intake was significantly decreased in the HFD-fed mice during the 8th week of HFD-feeding (F). (G) Intra-peritoneal glucose tolerance test (IPGTT) was completed during the 8 week HFD feeding period and was performed in mice that were fasted for 6 h. (H) AUC for the IPGTT was calculated from 0 to 120 min (units are millimoles per liter per minute). (I-K) TNF-α expression was downregulated while astrocytosis remained significantly upregulated at 8 weeks of HFD feeding in CD-1 mice. Nuclei are stained with DAPI (blue). Quantification of intensity of immunofluorescence (C, K) of CNTF, HSP70, GFAP and TNF-α in the ARC from C57BL/6 mice infused with saline or Intralipid (C), or from CD-1 mice fed either chow or HFD for 8 weeks (K), and quantification of the number of GFAP-positive astrocytes in the hypothalamic arcuate nucleus (D). The dashed boxes indicate the region used for quantification of intensity of CNTF, HSP70, GFAP and TNFα immunofluorescence, and GFAP-positive astrocyte number. Data in the bar graph are expressed as mean ± SEM (n = 5-6 animals/group); *P < 0.05 versus control. (TNF-α) and used as a template for real-time RT-PCR with primers specifically designed to amplify mRNA encoded by specific genes, n=3-4. The mRNA levels were quantified using the Ct method and normalized to the internal control Histone 3A. All results shown are relative to corresponding control mRNA levels and are expressed as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus control. Statistical analysis was performed using twoway ANOVA followed by post hoc analysis using Holm-Sidak t-test. Statistical analysis was performed using two-way ANOVA followed by post hoc analysis using Bonferroni test.

Figure 1 :

 1 Figure 1: Effect of acute Intralipid treatment at 24 h post-infusion in C57BL/6 mice on hypothalamic arcuate nucleus (ARC) reactive astrocytosis and expression of TNF-α, a marker of inflammation, and effect of high fat diet (HFD, 60% kcal) feeding for 8 weeks in CD-1 mice on body weight gain, glucose tolerance, ARC reactive astrocytosis and expression of TNF-α. (A, B, I, J) Representative images showing expression of immunofluorescence for GFAP (green) and TNF-α (red) in the hypothalamic ARC in C57BL6 mice at 24 h following acute infusion with saline (A) or Intralipid (B), or in CD-1 mice fed with chow (I) or HFD (J) for 8 weeks. (A-D) Inflammation in the ARC, indicated by significant upregulation of astrocytosis and TNF-α expression (A-D) without any change in CNTF or HSP70 expression (C) was evident within 24 h

Figure 2 :

 2 Figure 2: Effect of high fat diet (HFD, 60% kcal) feeding for 8 weeks in CD-1 mice on the markers of reactive astrocytosis, neuroprotective response to neuron injury, and proinflammatory and appetite regulating genes in the hypothalamic arcuate nucleus (ARC). (A-G) Representative images showing expression of immunofluorescence for GFAP (A, B, D, E), HSP70 (A, B), and

Figure 3 :

 3 Figure 3: Effect of high fat diet (HFD, 60% kcal) feeding for 20 weeks on mouse hypothalamic arcuate nucleus (ARC) neurons. (A-D) Representative images showing expression of doublelabelled immunofluorescence images for AgRP (green) (A, B) or NPY (green) (C, D) and TNF-α (red) (A-D) in the hypothalamic ARC following chow (A, C) or HFD (B, D) feeding for 20 weeks in CD-1 mice: HFD feeding induces hypothalamic inflammation as indicated by increased expression of TNF-α, a marker of inflammation, in the AgRP and NPY neurons of the ARC. (E) Graphical representation showing quantification of the number of neurons co-expressing AgRP or NPY and TNF-α in the ARC of CD-1 mice: HFD feeding significantly increases the number of AgRP and NPY neurons co-expressing TNF-α. The dashed boxes indicate the region used for quantification of intensity of AgRP, NPY, and TNF-α immunofluorescence, and TNF-α-positive AgRP and NPY neuronal number. (F) Representative images showing double-labelled immunofluorescence for TNF-α (red) and neuron-specific enolase (NSE, green) in the ARC of the hypothalamus. Nuclei are stained with DAPI (blue). Data in the bar graph are expressed as mean ± SEM (n = 5-6 animals/group); *P < 0.05 versus control.

Figure 4 :

 4 Figure 4: Gene expression analysis by real-time RT-PCR for the hypothalamic markers of inflammation, ER stress, neuroprotective response to neuron injury, and appetite regulation following (A,B) palmitate or (C,D) TNF- treatment in the hypothalamic NPY-expressing

Figure 5 :

 5 Figure 5: Effect of prolonged exposure of palmitate on NPY, AgRP, and CHOP mRNA levels in the hypothalamic mHypoE-44 and mHypoE-46 cell lines. The cells were treated with palmitate (50μM) and total RNA was isolated at the indicated time points over 33 h time period and used as a template for real-time RT-PCR with primers specifically designed to amplify mRNA encoded by specific genes. (A, B) NPY, (C, D) AgRP, and (E, F) CHOP mRNA levels were quantified using the Ct method and normalized to the internal control Histone 3A in the mHypoE-44 (A, C, E) and mHypoE-46 (B, D, F) neuronal cell lines, n = 3-4. All results shown are relative to corresponding control mRNA levels at each time point and are expressed as mean as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus control.

Figure 6 :

 6 Figure 6: (A-D) Effect of pre-treatment with the IKKβ inhibitor PS1145 on palmitate-mediated regulation of NPY and CHOP mRNA levels in the hypothalamic mHypoE-44 and mHypoE-46 cell lines. (E-J) Effect of pre-treatment with the IKKβ inhibitor PS1145 on TNF-α-mediated regulation of NPY, IκBα, and IL-6 mRNA levels in the hypothalamic mHypoA-NPY/GFP and mHypoE-46 cell lines. The cells were pre-treated with PS1145 (20 M) 1 h prior to treatment with palmitate (50 M) or TNF-α (50 ng/ml) and total RNA was isolated for at 4 h (palimitate) or 6 h (TNF-α) and used as a template for real-time RT-PCR. (A,B) NPY, (C,D) CHOP, (E,F) NPY, (G,H) IκBα, and (I,J) IL-6 mRNA levels were quantified using the Ct method and normalized to the internal control Histone 3A in the cell lines indicated, n = 3-4. All results shown are relative to corresponding control mRNA levels at each time point and are expressed as

  Figure 1

  Figure 4

Figure 5 ©

 5 Figure 5 © 2016 Macmillan Publishers Limited. All rights reserved.

  Figure 6DMSO

Acknowledgements

We acknowledge funding from the Canadian Institutes for Health Research (CIHR) (DDB, MBW), Canadian Diabetes Association (DDB, MBW), and Canada Research Chairs Program (DDB). Scholarship support through the Banting and Best Diabetes Research Centre (PSD, LW), CIHR Fellowship (LW), NSERC Studentships (PSD, DQT), and an Ontario Graduate Scholarship (PSD, DQT) is much appreciated.


Moses Eshovo Ojo

Fréderic Fauquet

Damien Bigourd

Patrick Mounaix

THz Spectroscopic Characterization of Oil Shales, IRMMW-THz 2022

come

THz Spectroscopic Characterization of Oil Shales, IRMMW-THz 2022

Moses Eshovo Ojo * , Fréderic Fauquet * , Damien Bigourd * , Patrick Mounaix * * Laboratoire IMS UMR CNRS 5218, Université de Bordeaux, France Abstract-Grey to dark-grey samples reported to be oil shales have been analyzed under the THz reflection and transmission spectroscopy. In order to examine the organic and mineral stratification of the oil shales, samples at varying height above the ground level were excavated from stream banks and hilly terrains. The constituent of these aquatic and terrestrial-based oil shales were further correlated to investigate any correspondence or dissimilarities in their composition by examining the refractive index, absorption and optical path length maps of the samples.

I. INTRODUCTION

il shale is a combustible source rock, composed of minerals and an organic matter referred to as kerogen. Oil shale is capable of generating oil and other petroleum products when subjected to pyrolysis. Shale oil and oil shale-gas which are among the pyrolytic products of oil shale are chemically identical to the products of fossil fuel, and thus serve as a substitute for conventional crude oil and natural gas respectively 1 . They hold important application in the context of a decline in the crude oil reserves and spike in the oil prices. Oil shale in widespread deposits has been reported in several countries. The oil shale samples in this work were sourced from Lokpanta, Nigeria.

Among its huge uses, THz-based spectroscopy has strong applicability in the development of the oil industry due to its non-destructive testing and strong interaction with organic matter. Conventional oil source rocks and petroleum products have well been investigated via the THz spectroscopy. In recent years, the investigation of oil shale via THz spectroscopy is gaining popularity. The kerogen constituent of the oil shale which is a delicate and complex heterogeneous organic substance making up a small percentage by mass of the total weight of the oil shale determines the quality and quantity of the oil yield. The vast majority THz spectroscopy experiments involve probing this organic content.

In a typical oil shale THz transmission spectroscopy at room temperature, THz pulses are mainly absorbed by the kerogen content, due to the fact, that the large macromolecules with aromatic compounds typically have vibration modes in the THz region around 0.5-4 THz. When the THz spectroscopy is performed with a pyrolyzed oil shale sample, there are generally two major phases in the optical response of the kerogen to the THz pulses 3 . The first phase occurring at lower temperature, involves the evaporation of moisture from the oil shale thereby resulting to an increase in the strength of the transmitted THz signal. The second phase is typically characterized by a decrease in the strength of the THz pulse, due to absorption of the pulses at high temperature. At such high temperature, the kerogen macromolecules decompose into lighter molecules mostly with short chains alkanes, while the minerals are decomposed into metals and metallic oxide. At this stage, the absorption of THz pulse is reversed, and mainly dominated by the metallic components. The responses of oil shales have been interpreted in terms of absorption, refractive index plots and contrast image maps. These above parameters have been shown to be related to the oil yield potentiality of the oil shale 2 . Fig. 1.0. THz spectroscopy reflection geometry (the incoming THz pulse is firstly reflected at the air-sample interface, giving rise to pulse 1 which was utilized by [START_REF] Miao | Simultaneous determination of organic distribution and content in oil shale by terahertz imaging[END_REF] to construct the subsurface image of oil shales. Pulse 2 which corresponds to the refracted pulse at the sample-air was used in our work. While pulse 3 is the reflected pulse from the metallic surface due to void space in-between the sample and metal surface.

A plot of refractive index , normally indicates a narrow decrease with frequency. In general, oil shales with lower , exhibits a negative correlation with oil yields, i.e. high-oil yielding oil shales have lower and vice versa. This is particularly valid for samples excavated along the bedding plane. While the absorption coefficient α, of independently analyzed oil shales have revealed that, higher corresponds to higher oil yield, and vice versa. Also, THz subsurface imaging of oil shales has been achieved via the reflection mode of the THz-based spectroscopy technique 3, 4 (refer to Fig. 1.0. In order to realize a robust information, we shall analyze the THz pulse 2 in our work). The amplitude of reflected THz pulse from different regions of the oil shale was employed to reconstruct the non-uniform distribution of organics on the oil shale surface. Three regions corresponding to high kerogen, low kerogen and mineral regions were observed based on the intensity distribution. Results from optical micrographs and micro-Raman O spectra showed good agreement with the THz contrast image. In our work, we aim to study the organic distribution of some reported oil shales in relation to elevation and environment type. The height of the undermined bank of streams measured about 11-16 m, while the lowland hills measured 5 -9 m.

II. RESULTS

The absorption map of four selected oil shale samples are shown for the purpose of a qualitative observation of the trend in kerogen abundance between the aquatic and terrestrially sourced oil shales. The THz pulses are absorbed strongly in the dark red regions, and mostly reflected at the bluish region. The thickness is about 1.85mm.

III. SUMMARY

We have used a combination of experimental data and numerical simulations to find parameters that best describe reflections and transmission from a given location in oil shale samples. The reflected terahertz time domain waveform is simulated using Fresnel's equations. Absorption and optical index images were analyzed to support the presence and the concentration of organic material within the samples.

Fig. 2 .

 2 Fig. 2.0. Stream bank oil shale located between 0-2m above the stream.The comparison between Fig.2.0 and Fig.3.0 indicates that oil shales at the base of the stream are richer in kerogen compared to shales farther from the stream beds.

Fig. 2 . 1 .

 21 Fig. 2.1. Stream bank oil shale located at 5m above the stream. The kerogen pattern of distribution follows a reverse trend among terrestrially sourced oil shale, as we observed from Fig. 3.0 and Fig. 3.1 that oil shale richer in kerogen are located at higher elevation from the foot of the hill. The light bluish region are probably occupied by minerals. The homogeneity of the sample in Fig. 1.0 was confirmed via the room-temperature based terahertz spectrum (with a baseline correction applied) as shown in Fig. 4.0. It was recorded with a Teraview Lx time-domain spectrometer in which three different locations on the sample were probed. Their index of refraction and absorption coefficient were found without significant dispersion.

Fig. 3 .

 3 Fig. 3.0. Hilly terrain oil shale located 2m above the foot of the hill.

Fig. 3 . 1 .

 31 Fig. 3.1. Hilly terrain oil shale located 5m above the foot of the hill.


M Chung

To Sang

L Garrigues
email: garrigues@laplace.univ-tlse.fr

T Dubois

S Tsikata
email: sedina.tsikata@cnrs-orleans.fr

3D sparse PIC modeling of a Hall thruster: Comparisons with experimental results IEPC

This work presents a new approach of the explicit electrostatic Particle-In-Cell (PIC) method. It uses the sparse grids as an alternative representation of the full grid domain. These grids are built with coarser anisotropic and isotropic grids through a combination technique. Change of basis allows to move from the sparse grid to the regular grid and vice versa. The method when integrated to the PIC scheme gives significant speed up in computational time by reducing the number of particles needed to reduce the statistical noise, and by making Poisson equation solved on coarser grids, especially for 3D simulations. Drawbacks of the method are that the combination technique introduces an additional grid approximation of the solution it reconstructs, so that the sparse grid and combination technique must be chosen wisely depending on the physics it aims to represent. Also, sparse grid methods are yet to be adapted to complex geometries and are limited to cubes in this work. As this work aims to compare numerical simulation with experimental data of a Hall thruster, adaptation of the method has been made to represent both the plume where experimental measures are taken and the channel of the Hall thruster. First results in transient state are presented for a 3D sparse PIC simulation, and computational time estimated to 20 days for fully converged simulation with electron density reaching 2𝒙10 18 𝒎 -3 . = Two-dimensional sub-grid of level I in horizontal direction and J in vertical direction

I. Nomenclature

II. Introduction

Standard electrostatic Particle-In-Cell (PIC) method uses a kinetic model to compute particle trajectory and a grid approach to solve the Poisson equation to obtain electric potential to the grid. Then electric field is computed to particle position by differentiating the electric potential and interpolation. This explicit approach introduces constraints of grid spacing and time step related to the resolution of electron properties (Debye length and inverse of plasma frequency). These inner limitations induce exponential dependence on dimension for numerical complexity and large number of particles to compute to reduce statistical noise which result in high computational time in three dimensional simulations. Such problems invite us to look into an alternative sparse PIC method exchanging a significant increase in computational time with an additional approximation over the grid potential.

III. Gains in Sparse PIC Method

The sparse PIC method defines a hierarchy of anisotropic and isotropic sub-grids with coarser mesh ([1] and [START_REF] Jacob | Efficient regular sparse grid hierarchization by a dynamic memory layout[END_REF]). It applies to initial grids with 2 𝑁 + 1 points in each direction of space, where N is the grid level, and thus are limited to simple geometry such as square in two dimensions and cubes in three dimensions, up to the author's knowledge. Charge deposition is done on these sub-grids as well as solving Poisson equation. The ratio between the total number of cells in a regular grid and the number of cells of these sub-grids reaches a factor 1000 when considering a 512 3 -cells three dimensional regular grid and scales as shown in Fig. 1 . It means that there are less operations to make for charge deposition as well as avoiding solving Poisson equation on fine grids, resulting in a direct gain in computational time and making the use of efficient direct solver possible. Less cells means also less particles if we keep the same number of particles per cell. Sparse grids have proven to be reducing statistical noise ( [START_REF] Muralikrishnan | Sparse grid-based adaptive noise reduction strategy for particle-in-cell schemes[END_REF]) and thus have a good synergy with PIC algorithm.

Fig. 1 Scaling of the ratio of regular grid number of cells over sparse method number of cells with the grid level N. M is the number of points in each direction for grid level N=9 and N=13.

After getting potential on these sub-grids, the coefficients expressed in the nodal basis are moved to the hierarchical basis thanks to a hierarchization algorithm. In this basis, an appropriate sum of the sub-grids, namely the combination technique, see example in Fig. 2, results in the construction of the sparse grid also called hierarchical grid. This first part of the algorithm is easily parallelised on OpenMP/MPI architecture. Though its principle is based on the cancellation of errors in the coarse grids solving, the combination technique used can be critical to the reconstruction of the solution as it adds an additional grid error. The choice of the appropriate sub-grids used for combination technique remains a topic of interest to capture the whole plasma physics in the Hall thruster and is also being investigated but let for future prospects in this work.

Then a dehierarchization algorithm transforms the hierarchical solution into the nodal solution on the regular grid, allowing to perform standard differentiation on the regular grid to compute electric field and interpolate it to the particle

Fig. 2 Example of combination technique in two dimensions for grid level N=2

position. Using this implementation allows to avoid interpolating electric field for all sub-grids which are dozens in number in three dimension; for instance, they are 64 for a 𝑁 = 7 grid. However it is more difficult to parallelize on OpenMP/MPI architecture, and other solution using GPU might be considered.

The rest of the algorithm is the same than standard PIC scheme. Particles are pushed with the Boris algorithm and no collisions are taken into account in this work. It is important to notice that the sparse PIC algorithm requires far less memory usage than the standard PIC algorithm, making memory requirements not critical.

IV. Towards comparison with experimental data

The experimental setup of the PIVOINE 2021 measurement campaign at ICARE, Orléans, calls for numerical simulation adaptation. Incoherent Thomson scattering measurements along radial and azimuthal directions of a PPS 1350 Hall thruster were made for Xenon and Krypton and could give the electron density, temperature and drift velocity profiles. Considering one measure needs 10 minutes of data acquisition (6000 laser pulses with an acquisition window of 10 nanoseconds every 0.1 second), simulation must have converged to give time averaged profiles.

Also, laser measurements catch photons coming from a small volume equivalent to a cylinder of 0.9 mm of diameter and 1.5 mm of length due to glass fibers and optics so that profiles must be space averaged.

Measurements being made out of the canal at 1 mm of distance from the exit, the plume must be part of the simulation domain. Specific functioning regime has been used for the campaign with 130V at the anode and a radial magnetic field gradient has been measured. This implies that the source term in the Hall thruster simulation is not uniform in the radial direction, which makes it difficult to model as it is often a fixed parameter of the simulation. Results of the experimental campaign are presented in [START_REF] Dubois | Insights into Hall thruster radial physics via incoherent Thomson scattering[END_REF] and tend to show a divergence of the plume with outer electron density and temperature being superior to inner one.

V. 3D sparse PIC computation of a Hall like thruster

All figures in this section are presented in Appendix: Figures for clarity, with international unit system. The simulation domain used for this preliminary work is being represented in Fig. 3 and4. Key simulation parameters are presented in Table . 1. Initial density profiles are uniform for both ions and electrons. For each ion leaving the domain, we inject an electron-ion pair with cosine distribution in the ionization zone. A neutralization current equal to the difference between the electron and ion current at the anode is injected close to the cathode like in [START_REF] Charoy | 2D axial-azimuthal particle-in-cell benchmark for low-temperature partially magnetized plasmas[END_REF]. We impose periodic conditions in the azimuthal direction and Dirichlet conditions in the others.

Figures 5,6,7 and 8 show transient state of the Hall thruster discharge at one microsecond obtained with 3D sparse PIC simulation for ion density profile and the azimuthal electric field associated in the axial azimuthal plane, and axial electric field and potential in the axial radial plane. Comparison with other codes ( [START_REF] Charoy | 2D axial-azimuthal particle-in-cell benchmark for low-temperature partially magnetized plasmas[END_REF] and [START_REF] Villafana | 3D Particle-In-Cell modeling of anomalous electron transport driven by the Electron Drift Instability in Hall thrusters[END_REF]) fully converged ExB Hall thruster discharges will be performed in future work.

VI. Conclusion

This work has presented the Sparse PIC method adapted to a three dimensional Hall thruster model and shown qualitative figures in transient state. As a recent numerical technique, many questions still remain. We address several points of discussion and considerations: 1) Gains in computational time depend on level of sparse grids with the current optimization and domain (3.8s/dt for N=9, 0.46s/dt for N=8 , 0.08s/dt for N=7 with 1440 cpus shared between 80 sockets). Charge deposition costs more than 75% of the computational time for N<=7 and remains important for N>=8 as the number of sub-grids grows too. 2) Sparse cubes used as elementary brick to represent some types of geometries have slight effects at cube frontiers and this alternative method needs investigation. It also needs to be adapted to more complex geometries, such as the curved walls of the Hall thruster. 3) MPI/OpenMP architecture might not be relevant for dehierarchization algorithm which reconstructs potential on the regular grid for all sockets to avoid expensive MPI communications. 4) The model of the Hall like thruster will be completed for taking into account non-uniform radial ionization, and investigation over electron secondary emission effects will be pursued. 5) Use of different combination techniques can be investigated to get more precision in the results, especially with truncated/offset approaches ( [START_REF] Deluzet | SPARSE GRID RECONSTRUCTIONS FOR PARTICLE-IN-CELL METHODS[END_REF]). We would like to emphasize that this work aims to compare numerical Sparse PIC simulation with experimental data and that any comparison with other PIC simulation is welcomed.

Appendix: Simulation Figures

Fig. 3 Fig. 6 6 Fig. 7

 3667 Fig. 3 Simulation domain -2D axial/radial cut

Fig. 8

 8 Fig. 8 Potential 2D axial-radial profile.

Table 1 Key simulation parameters.

 1

	Grid level	8
	Simulation domain	4.5𝑥1.5𝑥3𝑐𝑚 3 -four cubes of 1.5𝑥1.5𝑥1.5𝑐𝑚 3
	Channel	1.5𝑥1.5𝑥1.5𝑐𝑚 3
	Plume	4.5𝑥1.5𝑥1.5𝑐𝑚 3
	Source term	z=0.93cm -1.5cm. Cosine both directions radial x and axis z
	Axis-position of cathode electron injection	2.9cm
	Anode potential	200V
	Cathode potential potential	0V
	Magnetic field maximum at the center of the channel 100G
	Initial density	5𝑥10 16 𝑚 -3
	Time step	5𝑥10 -12 𝑠
	Final time	1𝑥10 -6 𝑠
	Computational time	1 day and 1 hour

Acknowledgments

This work was supported by the ENS Paris-Saclay Doctoral Grant Program (MESRI funding). This work was granted access to the HPC resources of CALMIP supercomputing center under the allocation 2013-P1125.


N De Mejanes

R Pascaud

V Mazières

L Garrigues

O Pascal

V Laquerbe

A Rossi

Simulation of the interaction between Hall thruster and communication system integrated on a small spacecraft IEPC

Simulation of the interaction between Hall thruster and communication system integrated on a small spacecraft N de Mejanes, R Pascaud, V Mazières, L Garrigues, O Pascal, V Laquerbe,

I. Introduction

The miniaturization of satellites reduces the cost of putting them into orbit. As access to space becomes more affordable, private projects increase and more and more CubeSats are being launched. However, we have little experience of the technologies that enable miniaturization, particularly for the propulsion system and its integration. The use of electric propulsion has made it possible to significantly reduce the size and weight of the thrusters [START_REF] Levchenko | Space micropropulsion systems for Cubesats and small satellites: From proximate targets to furthermost frontiers[END_REF]. However, the integration of these systems raises new issues, particularly with regard to electromagnetic compatibility [START_REF] Holste | Ion thrusters for electric propulsion: Scientific issues developing a niche technology into a game changer[END_REF]. Indeed, the plume can be source of electromagnetic interference with other subsystems such as the communication one and its microwave antennas. These issues have been studied in the past on large platforms. Two effects due to the presence of the plume have been identified: the emission of RF noise in the microwave antennas bandwith and the modification of the propagation channel [START_REF] Beiting | Time-Domain Characteristics of 0.5 -8 GHz Pulsed Emission from Hall Thrusters[END_REF][START_REF] Bandinelli | EM Modelling of RF propagation through plasma plumes[END_REF]. As a result of these studies, it was suggested that this problem should be addressed by an integration response, i.e., moving the thruster as far away as possible from the microwave antennas to avoid any problem. However, as illustrated in Fig. ref, this solution is no longer possible on small platforms due to the proximity of all the subsystems. The antennas can no longer be isolated from the thruster and its plume. In this work, we propose a numerical method to represent the propagation of signals emitted by microwave antennas located near the plume of a Hall thruster, which is a good candidate in a miniaturization context.

II. Numerical method

An overview of the proposed method is shown in Fig. 2. The plume, which is an inhomogeneous and dispersive medium, is represented by a set of materials of different relative permittivity. The local permittivity is calculated using the Drude model which relates the local electron density within the plume to an equivalent relative permittivity. The numerical analysis of the integrated microwave antennas, with and without the plume, is finally performed with an electromagnetic solver. In the following subsections, we detail each of these steps.

A. Electron density

Electron density within the plume in the input data required in order to treat this problem. This distribution depends on the thruster type and its operating condition. There are many different technologies, and they do not exhaust the same plume in terms of composition, density, temperature, or shape. In addition, some of these thrusters operate in a pulsed regime leading to a pulsed behaviour of the plume and a more complex study of the interaction with a microwave signal. Even for so-called stationary thrusters, there are instabilities that lead to oscillations in the plasma electron density [START_REF] Mazouffre | Electric propulsion for satellites and spacecraft: established technologies and novel approaches[END_REF]. Although a spatio-temporal description would be required to describe the effect of the plasma on the propagation of microwave signals, in this paper, as a first approximation, the plume will be considered stationary. The study of the stationary plume will highlight the degradation of the communication link, but it is important to stress that temporal modulations of the plasma could further degrade the microwave communication link.

In this work, as an example, we propose to study the plume of a low power Hall thruster, which is a good candidate in a miniaturization context. Here, the JET2D simulation code was used [START_REF] Garrigues | Modeling of the plasma jet of a stationary plasma thruster[END_REF]. JET2D is based on a plasma jet model of a Hall thruster. It is a hybrid model where the ions are processed in a particular way by the Monte Carlo particle in cell (PIC) model and the electrons are processed in a fluid way by the local electron density linked to the local potential by Boltzmann's law. The electric field is recalculated at each time step by assuming quasi neutrality. The potential is then deduced from Boltzman's law. JET2D was originally developed and experimentally validated for standard SPT100 type Hall thrusters used on satellites larger than those of our interest. Thanks to the JET2D input parameters like gas flow rate, specific impulse, discharge current, ion current to total current ratio, and ejection angle we can still approach the conditions of a representative plume of a micropropulsion Hall thruster. Therefore, the input parameters of JET2D, as the angular distribution, have been optimized so that the simulated current density is the closest to the measured current density of 100 W class Hall thruster [START_REF] Hallouin | Far-Field Plume Characterization of a 100-W Class Hall Thruster[END_REF]. Specific impulse 𝐼 𝑠 𝑝 , gas flow rate 𝑚 𝑋𝑒 , and discharge current 𝐼 𝑑 have been fixed as in Ref. [START_REF] Hallouin | Far-Field Plume Characterization of a 100-W Class Hall Thruster[END_REF], namely 𝐼 𝑠 𝑝 = 1740 s, 𝑚 𝑋𝑒 = 0.50 mg/s, and 𝐼 𝑑 = 0.44 A. Fig. 3 shows the distribution in a half plane of the electron density obtained from JET2D for the parameters described previsously. The origin 𝑥 = 0 cm and 𝑦 = 0 cm represents the center of the half Hall thruster. The electron density is maximum at the thruster exit and decreases along the 𝑥 and 𝑦 directions. On the simulation domain, the electron density varies between 𝑛 𝑒 = 6.86 × 10 10 cm -3 close to the thruster exit and 𝑛 𝑒 = 5.30 × 10 5 cm -3 far from it. The electron density gradient is strong especially in the 𝑦 direction, indeed for 𝑦 = 0 cm and 𝑦 = 20 cm in the plane of the thruster exit, the electron density is respectively equal to 𝑛 𝑒 = 6.86 × 10 10 cm -3 and 𝑛 𝑒 = 2.6 × 10 7 cm -3 , i.e., a decrease higher than three orders of magnitude.

B. Drude model

From the microwave point of view, the Hall thruster plume simulated with JET2D is represented as an isotropic dispersive medium with a complex equivalent local relative permittivity given by the Drude model [START_REF] Lieberman | Principles of Plasma Discharges and Materials Processing[END_REF]

𝜖 𝑝 (ì 𝑟) = 1 - 𝜔 2 𝑝,𝑒 (ì 𝑟) 𝜔(𝜔 -𝑗 𝜈 𝑒-𝑛 ) (1)
with 𝜔 the angular frequency of the wave that interacts with the plasma, 𝜈 𝑒-𝑛 the electron-neutral collision frequency, and 𝜔 𝑝,𝑒 (ì 𝑟) = √

𝑛 𝑒 ( ì 𝑟)𝑒 2
𝑚 𝑒 𝜖 0 the angular plasma frequency where 𝑛 𝑒 (ì 𝑟) is the local electron density, 𝑒 the elementary charge, 𝑚 𝑒 the electron mass, and 𝜖 0 the vacuum permittivity.

The real part of the relative permittivity represents its dielectric constant and the imaginary part the energy absorption via the electron-neutral collision frequency. For a plume, the collision frequency which is proportional to the neutral density, is very low compared to the frequency of the microwave signal since the neutral pressure is very low in space vacuum condition. For a neutral density on the order of 10 12 cm -3 and an electron temperature of 2 eV and the electron-neutral collision frequency is around 3 × 10 5 s -1 . So the ratio between the collision frequency and the frequency of a microwave signal is very low and the plasma is therefore weakly collisional. The imaginary part is then negligible compared to the real part of the permittivity (i.e.. Fig. 4 shows the distribution of the real part of the relative permittivity for a frequency of 436 MHz for the electron density showed in Fig. 3. We can see a zone where the real part of the permittivity is negative. In this area, electromagnetic waves become evanescent and therefore do not propagate. We also notice a gradient in the dielectric constant and so in the index of refraction 𝑛 = √ 𝜖 𝑝 all along the plume.

C. EM solver

Finally, in order to study the propagation of the microwave signal emitted or received by onboard microwave antennas through the plume of a thruster integrated on small satellite, we use a commercial electromagnetic numerical code that solves Maxwell's equations. More specifically, we use Ansys HFSS that is based on the finite element method (FEM) to solve time-harmonic Maxwell's equations. This code provides the far field characteristics of the antenna studied with and without plasma. For this purpose, we first present the method for plume discretization.

It is well known that the dielectric permittivity of a plume is continuous and inhomogeneous, but it must be discretized in the solver to be simulated. Although the plume is already sampled in the JET2D model, it is necessary to choose a discretization scheme adapted to our electromagnetic interaction problem. Indeed, the discretization needed to represent the plume physics is not the same to represent the plume/microwave interaction.

It was chosen to represent the plume by an assembly of toroids. Indeed, the electron density distribution obtained from JET2D consists of a 2D grid where in each grid cell the local electron density is given. Each grid cell in 2D is equivalent to a 3D toroidal object using axial symmetry along x direction. In each of these objects the local relative permittivity is fixed using the electron density of the cell in question and the Drude model. The axial and radial resolution Δ𝑥, Δ𝑦 of each square section toroid is a function of the wavelength 𝜆 of the signal such that Δ𝑥, Δ𝑦 ≪ 𝜆 and with the aim of limiting the simulation time. In this study for a frequency equal to 436 MHz, Δ𝑥 and Δ𝑦 are both equal to 1 cm so the ratio Δ𝑥, 𝑦/𝜆 = 0.0145.

Another parameter to set is the maximum limit of the relative permittivity, that is to say the limit between what we consider to be the plasma plume and vacuum in the simulation. In other words, we need to specify a given threshold from which any element coming from JET2D will be simply represented by vacuum and not as plasma in the electromagnetic solver. The objective is to limit the computational time because the more elements to simulate, the longer it takes. But we still need to represent correctly the problem so that this choice does not influence the final results. In this aim, a convergence study (not shown here) has been performed in order to choose the value of the limit permittivity, which is in our case 𝜖 𝑝 = 0.95. Fig. 5 shows the simulated and discretized plume when considering these choices, i.e, a limiting permittivity of 0.95 and a spatial resolution of 1 cm. The plume under these conditions is 71 cm long and 54 cm wide. The left side of the Fig 5 shows permittivity with the limit permittivity contour line equal to 𝜖 𝑝 = 0.95. The relative permittivity ranges from -28 in highest density area near the thruster exit plane to a maximum of 0.95. Beyond this, the medium is considered to be vacuum. The right-hand side of the figure shows the discretization scheme used to construct the torroids in the electromagnetic solver. Here, the plume is the assembly of 1097 torroids of different relative permittivities.

III. Results

In this section the characteristics of a 436 MHz dipole antenna is studied with the Hall thruster plume and the discretization conditions described before. Fig. 6 shows the representation of the whole problem in the electromagnetic solver. The antenna is positioned on the center of the top face of a 6U, i.e. a platform with dimensions equal to 10 × 20 × 30 cm 3 . The discretized plume is in the centre of the lower face to represent a thruster at this place. Also the two main cut planes for the radiation pattern of this antenna, namely 𝜙 = 0 • and 90 • are also shown in Fig. 6. Fig. 7 compares the real and imaginary part of the input impedance 𝑍 of the antenna on 406 -466 MHz band when the thruster is OFF (simulation without the plume) and when the thruster is ON (simulation with the plume). We observe that the presence of the plume has little or no influence. In both cases, for a frequency equal 436 MHz, the real and imaginary part are close to 𝑅𝑒(𝑍) = 50 Ω and 𝐼𝑚(𝑍) = 0 Ω. Thus, whether the plume is present or not, the antenna still appears to matched to 50Ω at 436 MHz.

Fig. 8 shows the simulated gain radiation pattern, i.e., the angular energy distribution of the 436 MHz dipole as a function of angle 𝜃 in the 𝜙 = 0 • , 90 • planes for the ON and OFF conditions for the thruster. In both planes, a decrease in gain equal to 1 dB is observed for angles 𝜃 close to 180 • , i.e., around the plume position. Since there is no loss in the plasma due to the non-collisional assumption, the energy is not absorbed by the plasma. The decrease in gain in this area is due to a change in the directivity pattern of the antenna since the plume, due to its permittivity gradient, behaves as a refracting medium which can change the far field radiation characteristics of the antenna.

IV. Conclusion

The proposed method provides a simple way to evaluate the interaction between an electric thruster plume and integrated microwave antennas on a CubeSat platform. The only input data required is the distribution of the density distribution within the plume. In this way, a wide variety of cases could be tested: several types of thrusters, operating conditions, antennas, or integration. In order to validate the proposed method, this work will soon be completed by measurements of the far field radiation of a microwave antenna in the presence of plasma in an anechoic chamber.

Fig. 1

 1 Fig. 1 Representation of a large satellite (Eutelsat 172B from Airbus Defense and Space ©) next to a 6U CubeSat.

Fig. 2

 2 Fig. 2 Numerical method overview.

Fig. 3

 3 Fig. 3 Half plane distribution of the electron density in the Hall thruster plume simulated with JET2D.

Fig. 4

 4 Fig. 4 Half plane distribution of the relative permittivity for a frequency of 436 MHz.

Fig. 5 436

 5 Fig. 5 436 MHz plume permittivity contour lines (left) and discretization scheme (right).

Fig. 7 Fig. 8

 78 Fig. 7 Real and imaginary part of the impedance (𝑍) as a function of the frequency with (ON) and without (OFF) the plasma.

naomi.de-mejanes@isae-supaero.fr

romain.pascaud@isae-supaero.fr

Acknowledgments

The authors would like to thank the Centre National d'Etudes Spatiales (CNES), and the Institut Supérieur de l'Aéronautique et de l'Espace (ISAE-SUPAERO) for their financial support.