GINGER ROOT EXTRACT FOR NEUROPATHIC PAIN

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1025432
• Grant No.: 2021-67017-34026
• Cumulative Award Amt.: $500,000.00
• Proposal No.: 2020-04545
• Project Start Date: Jan 1, 2021
• Project End Date: Dec 31, 2024
• Grant Year: 2021
• Program Code: [A1343]- Food and Human Health

Project Director
Shen, C.

Recipient Organization
TEXAS TECH UNIVERSITY HEALTH SCIENCES CENTER
3601 4TH STREET
LUBBOCK,TX 794300002

Performing Department
Pathology Dept Lbk

Non Technical Summary
Pain is a major health care problem and the number one reason for patients to seek medical help. Pain serves a useful warning function, but for reasons not well understood, pain can persist and develop into a complex syndrome. Persistent or chronic pain is associated with negative emotional states such as anxiety and depression and with cognitive deficits. Pain as a complex disorder is still difficult to treat because underlying mechanisms are not well understood.Ginger is a well-known spice plant. Ginger can have analgesic and anti-inflammatory effects. It has been used for a variety of pain-associated ailments such as osteoarthritis and opiate withdrawal-induced disorders. However, the effect of ginger in neuropathic pain is unclear. In this study, we would like to test the effects of ginger root extract supplemented into the diet on pain behaviors of rats in a neuropathic pain model. Two weeks after induction of the pain model, the animals will be assigned to one of four groups. They will receive no or 3 different doses of ginger root extract for 5 weeks. All animals will be tested in behavioral assays for different aspects of neuropathic pain. At the end of the experiment, we will collect feces for gut microbiome and metabolites markers as well as the tissues for the analysis of neuro-inflammatory markers.

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

0%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
701	2235	1010	100%

Knowledge Area
701 - Nutrient Composition of Food;

Subject Of Investigation
2235 - Herbs and spices;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

nutrition

gut

microbiota

pain

Goals / Objectives
The goal of thisstudy is to evaluate the effects of gingerols-enriched ginger (GEG) on neuropathic pain (NP)-associated behaviors in a preclinical animal model of NP and to elucidate underlying mechanisms. By applying the SD rat model of spinal nerve ligation (SNL)-induced neuropathy, we will evaluate the dose-dependent effects of GEG on pain-related behaviors and alteration of biomarkers in biological samples. Four specific aims are designed: 1) to measure the effects of GEG on NP-associated sensory and affective behaviors; 2) to determine the effects of GEG on gut health; 3) to determine the effects of GEG on neuroinflammation; and 4) to explore correlation between gut health and neuro axis in NP status due to GEG.We will usemaleandfemalerats toevalaute ifGEG has gender's effect on NP.

Project Methods
Project: A total of 60 male SD rats will randomly divided into 5 groups (n=12 per group): Sham, spinal nerve ligation (SNL) group, SNL+0.375% GEG (wt/wt diet) group, SNL+0.75% GEG (wt/wt diet), and SNL+1.125% (wt/wt diet) for 5 weeks. We will conduct pain-related behavioral assays including mechanosensitivity, vocalization and affective behaviors (conditioned place preference/ avoidance, open field test, and elevated plus maze); measure gut health-related parameters (gut microbiota, metabolites, intestinal integrity and inflammation); conduct biochemical assays on microglia activation, mitochondrial function, and pro-inflammatory cytokine/chemokine signaling in collected specimen (brain, dorsal root ganglia, spinal cord, and plasma); and perform correlation analyses among all outcome measures. Same experimental design and outcome measures will be applied to 60 female rats in a GEG-dose dependent manner.Statistical analysis: All averaged values will be presented as the mean±SEM. Statistical significance will be accepted at the level p<0.05. SPSS statistics will be used for all statistical analyses. Student's t-test will used to compare two sets of data that have Gaussian distribution and similar variances. For multiple comparisons, one-way or two-way ANOVA (repeated measures where appropriate) will be used with Bonferroni corrections for post hoc tests to compare difference between groups. In terms of microbiome data, after reads being filtered, denoised, and merged, we will use DADA2 plugin within QIIME2 to determine exact amplicon sequence variants (ASVs). Relative abundance of individual taxa and alpha diversity will be compared among groups using the non-parametric test Mann-Whitney U Test (Wilcoxon Rank Sum Test) and was regarded as significant when P < 0.05. We will employ the PICRUSt2 tool to investigate the functional profiles of the gut microbiome with different dietary treatments. The inferred functional profiles were based on the MetaCyc pathway ontology. Statistical analysis using Welch's t-test and was regarded as significant when p<0.05. Regarding analyses of metabolites, peak areas will be measured by compound discoverer (3.1) software and t-test will be performed to determine the number of statistically different metabolites in the SNL group compared to the other groups. PCA will be performed to assess the different profiling of the metabolites detected and quantified in all groups. In addition, an R package named weighted gene co-expression network analysis (WGCNA) will be applied to analyze the relationship between microbiomes and metabolomic (metabolites) data. Finally, Pearson or Spearman correlation analysis will be used to examine the correlations between NP-behavioral outcomes, gut health parameters, and neuroinflammation markers.Efforts: Laboratory instruction and learning opportunities for postdoc fellows and student assistants.Evaluation:Successful completion of the proposed study will provide strong scientific evidence on how GEG would mitigate NP-associated behavioral outcomes along with supportive gut microbiome/metabolites and biochemical outcomes. We plan to publish laboratory data in high impact peer-referred journals (at least 4 papers). We will also present the results at national/international scientific conferences in the fields of health, nutrition, pain, and agriculture as well as at USDA PD conferences.

Progress 01/01/24 to 12/31/24

Outputs
Target Audience:During the period covered by this 4th annual report, this project has provided research experience, experiential learning opportunities, and laboratory instruction to two research associates and 2 undergraduate students the Undergraduate Research Scholar Program and the Pi2 Program, respectively. Changes/Problems:There were no major changes in approved protocols during the reporting period. What opportunities for training and professional development has the project provided?We provided training in molecular biology, data analysis, interpretation and presentation to two senior research associates and the students for their professional development in the field of bioactive compounds in pain research. How have the results been disseminated to communities of interest?We published our work in 6 peer-referred journals in the area of bioactive compounds for neuropathic pain. Santos JM, Deshmukh H, Elmassry MM, Yakhnitsa V, Ji G, Kiritoshi T, Presto P, Antenucci N, Liu X, Neugebauer V, Shen CL (corresponding author). Beneficial effects of ginger extract on pain behaviors, inflammation and mitochondrial function in the colon and different brain regions of male and female neuropathic rats: a gut-brain axis. Nutrients. 2024; 16(20): 3563. doi: 10.3390/nu16203563. Shen CL, Deshmukh H, Santos JM, Elmassry MM, Presto P, Driver D, Bhakta V, Yakhnitsa V, Kiritoshi T, Ji G, Lovett J, Hamood A, Neugebauer V. Fecal microbiota transplantation modulates gut microbiome composition and glial signaling in brain and colon of rats with neuropathic pain: evidence for microbiota-gut-brain axis. Journal of Frailty & Aging. 2024. https://doi.org/10.14283/jfa.2024.65 Shen CL, Santos JM, Elmassry MM, Bhakta V, Driver Z, Ji G, Yakhnitsa V, Kiritoshi T, Lovett J, Hamood AN, Sang S, Neugebauer V. Ginger polyphenols reverse molecular signature of amygdala neuroimmune signaling and modulate microbiome in male rats with neuropathic pain: Evidence for microbiota-gut-brain axis. Antioxidants. 2024, 13, 502. https://doi.org/10.3390/ antiox13050502 Appell C, Jiwan NC, Shen CL, Luk HY. Curcumin mitigates muscle atrophy potentially by attenuating calcium signaling and inflammation in a spinal nerve ligation model. Current Issues in Molecular Biology.2024;46(11), 12497 - 12511;https://doi.org/10.3390/cimb46110742 Shen CL, Wang R, Santos JM, Elmassry MM, Stephens E, Neugebauer V. Ginger alleviates mechanical hypersensitivity and anxio-depressive behavior in rats with diabetic neuropathy through beneficial actions on gut microbiome composition, mitochondria and neuroimmune cells of colon and spinal cord. Nutrition Research. 2024; 124, 73 - 84. Doi: 10.1016/j.nutres.2024.01.014. Appell CR, Jiwan NC, Wang R, Shen CL, Luk HY. Ginger Supplementation Attenuated Mitochondrial Fusion and Improved Skeletal Muscle Size in Type 2 Diabetic Rats. In Vivo. 2024; 38(1): 73 - 81. doi: 10.21873/invivo.13412. What do you plan to do during the next reporting period to accomplish the goals?This is the 4th year (last year) annual report of our award, and we are happy to say that we have accomplished the goals proposed in this project. Thus, there will not be another next reporting period.

Impacts
What was accomplished under these goals? We have published 3 peer-reviewed articles. Here are the main findings: Santos et al. doi: 10.3390/nu16203563. GEG supplementation mitigated spontaneous pain in both male and female rats with NP while decreasing emotional-affective responses only in male NP rats. GEG supplementation increased intestinal integrity (claudin-3) and suppressed neuroinflammation [glial activation (GFAP, CD11b, IBA1) and inflammation (TNFα, NFkB, IL1β)] in the selected brain regions and colon of male and female NP rats. GEG supplementation improved mitochondrial homeostasis [increased biogenesis (TFAM, PGC1α), increased fission (FIS, DRP1), decreased fusion (MFN2, MFN1) and mitophagy (PINK1), and increased Complex III] in the selected brain regions and colon in both sexes. Some GEG dose response effects in gene expression were observed in NP rats of both sexes. Conclusion: GEG supplementation decreased emotional-affective pain behaviors of males and females via improving gut integrity, suppressing neuroinflammation and improving mitochondrial homeostasis in the amygdala, frontal cortex, hippocampus, and colon in the both male and female SNL rats in an NP model, implicating the gut-brain-axis in NP. Sex differences observed in the vocalizations assay may suggest different mechanisms of evoked NP responses in females. Shen et al. https://doi.org/10.14283/jfa.2024.65. After two weeks, the FMT of healthy gut microbiota decreased mechanical hypersensitivity in SNL rats [SNL+(Sham-FMT) vs. SNL+V]. A temporal shift in microbiome profiles after 2-week FMT treatment was observed in Sham+(SNL-FMT) and SNL+(Sham-FMT) groups, while the microbiome profile shifted back a certain extent after FMT ceased. At the end of study, the Sham+(SNL-FMT) group acquired low abundance of UCG-001, Odoribacter, and Peptococcaceae, and high abundance of UBA1819 and Victivallis. The SNL+(Sham-FMT) group maintained high abundance of Butyricimonas and Escherichia-Shigella. The SNL+(Sham-FMT) group had altered glial and macrophage activation/inflammation markers in the brain/colon than the SNL+V group. Relative to the SNL+V group, the SNL+(Sham-FMT) group had significantly lower gene expressions of GFAP (hypothalamus), IBA-1 (colon), and NF-kB (amygdala/colon), but higher gene expressions of complex I (amygdala/hypothalamus) and claudin-3 (amygdala/hypothalamus/colon). In conclusion, FMT containing healthy microbiota given to SNL rats attenuates mechanical hypersensitivity, modulates microbiota composition, and mitigates downstream glial activation/inflammation markers in a NP model. Shen et al. https://doi.org/10.3390/ antiox13050502. Both GEG (200 and 600 mg/kg) groups mitigated SNL-induced NP behavior. GEG-supplemented animals had a decreased abundance of Rikenella, Muribaculaceae, Clostridia UCG-014, Mucispirillum schaedleri, RF39, Acetatifactor, and Clostridia UCG-009, while they had an increased abundance of Flavonifactor, Hungatella, Anaerofustis stercorihominis, and Clostridium innocuum group. Relative to sham rats, Fos and Gadd45g genes were upregulated, while Igf1, Ccl2, Hadc2, Rtn4rl1, Nfkb2, Gpr84, Pik3cg, and Abcc8 genes were downregulated in SNL rats. Compared to the SNL group, the GEG200 group and GEG600 group had increases/decreases in 16 (10/6) genes and 11 (1/10) genes, respectively. GEG downregulated Fos and Gadd45g genes and upregulated Hdac2 genes in the amygdala. In summary, GEG alleviates NP by modulating the gut microbiome and reversing a molecular neuroimmune signature.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Shen CL, Deshmukh H, Santos JM, Elmassry MM, Presto P, Driver D, Bhakta V, Yakhnitsa V, Kiritoshi T, Ji G, Lovett J, Hamood A, Neugebauer V. Fecal microbiota transplantation modulates gut microbiome composition and glial signaling in brain and colon of rats with neuropathic pain: evidence for microbiota-gut-brain axis. Journal of Frailty & Aging. 2024. https://doi.org/10.14283/jfa.2024.65
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Santos JM, Deshmukh H, Elmassry MM, Yakhnitsa V, Ji G, Kiritoshi T, Presto P, Antenucci N, Liu X, Neugebauer V, Shen CL (corresponding author). Beneficial effects of ginger extract on pain behaviors, inflammation and mitochondrial function in the colon and different brain regions of male and female neuropathic rats: a gut-brain axis. Nutrients. 2024; 16(20): 3563. doi: 10.3390/nu16203563.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Shen CL, Santos JM, Elmassry MM, Bhakta V, Driver Z, Ji G, Yakhnitsa V, Kiritoshi T, Lovett J, Hamood AN, Sang S, Neugebauer V. Ginger polyphenols reverse molecular signature of amygdala neuroimmune signaling and modulate microbiome in male rats with neuropathic pain: Evidence for microbiota-gut-brain axis. Antioxidants. 2024, 13, 502. https://doi.org/10.3390/ antiox13050502
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Appell C, Jiwan NC, Shen CL, Luk HY. Curcumin mitigates muscle atrophy potentially by attenuating calcium signaling and inflammation in a spinal nerve ligation model. Current Issues in Molecular Biology. 2024; 46(11), 12497 - 12511; https://doi.org/10.3390/cimb46110742
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Shen CL, Wang R, Santos JM, Elmassry MM, Stephens E, Neugebauer V. Ginger alleviates mechanical hypersensitivity and anxio-depressive behavior in rats with diabetic neuropathy through beneficial actions on gut microbiome composition, mitochondria and neuroimmune cells of colon and spinal cord. Nutrition Research. 2024; 124, 73 - 84. Doi: 10.1016/j.nutres.2024.01.014.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Appell CR, Jiwan NC, Wang R, Shen CL, Luk HY. Ginger Supplementation Attenuated Mitochondrial Fusion and Improved Skeletal Muscle Size in Type 2 Diabetic Rats. In Vivo. 2024; 38(1): 73 - 81. doi: 10.21873/invivo.13412.

Progress 01/01/21 to 12/31/24

Outputs
Target Audience:During the whole award period covered by this final report, this project has provided research experience, experiential learning opportunities, and laboratory instruction to 5 research associates, 2 medical students in the Medical Student Research Program, and 3 undergraduate students through the Undergraduate Research Scholar Program and the Pi2 Program. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We provided training in animal handling, animal behavior, surgery, molecular biology, systems physiology, data analysis, interpretation and presentation to two senior research associates and the students for their professional development in the field of bioactive compounds in pain research. Through this project, we have provided opportunities for professional development to 5 research associates, 2 medical students, and 3 undergraduate students. Among them, one research associate (Julianna Santos) obtained a faculty position as an Assistant Professor of Microanatomy and Cellular Biology, Texas Tech University Health Sciences Center, El Paso, TX, one research associate (Moamen Elmassry) hasapplied for a faculty position nationwide, two medical students started their residency programs (Emily Stephens, MD, PGY1, Internal Medicine, Massachusetts General Hospital, Boston, MA; Carina Watson, MD, PGY1, Ob/Gyn, Houston Methodist Hospital, Houston, TX), one undergraduate student (Zarek Driver) was accepted into medical schoolat the Texas Tech University Health Sciences Center, Lubbock, TX, and two other undergraduates (Viren Bhakta and Jessica Contreras) have applied for medical schools. How have the results been disseminated to communities of interest?Through this project period, we have published 10 peer-reviewed journals in the area of bioactive compounds for neuropathic pain, presented 19 posters at local, national, and international conferences, and gave 4 invited oral presentations at local, national and international conferences. Overall, ginger extract actions on pain behavior through beneficial effects on the gut microbiome and metabolites, on neuroinflammation in the peripheral and central nervous system, and on neuropathology in a brain region important for the emotional-affective aspects of pain and pain modulation (amygdala). The project identified GEG as a useful tool to mitigate chronic pain through a mechanism that involves the gut-brain axis. What do you plan to do during the next reporting period to accomplish the goals?This is the final report of our award, and we are happy to say that we have accomplished the goals proposed in this project. Thus, there will not be another next reporting period.

Impacts
What was accomplished under these goals? Data collected:The following data were collected in 5 groups of male and female animals, which included sham controls and neuropathic rats treated either with vehicle or with different doses of ginger extract (200, 400, and 600 mg/kg body weight) for 4 weeks. The well-established spinal nerve ligation (SNL) model of neuropathic pain was used. In Aim 1, we collected pain-associated behavioral data (hypersensitivity, spontaneous pain, emotional responses, and anxiety-like behavior). In Aim 2, we collected gut health data (markers of intestinal barrier integrity,gut microbiome, and fecal metabolites). In Aim 3, we collected gene expression data of markers for neuroinflammation and mitochondrial homeostasis in the gut (colon) and peripheral (dorsal root ganglion) and central (spinal cord and different brain regions) nervous system. In Aim 4, we collected data of correlation analyses between fecal metabolites and pain as well as between neuroinflammation/mitochondrial gene profiles and gut microbiome composition. Summary statistics and discussion of results: Ginger extract alleviated pain-associated behaviors via mitigation of neuroinflammation In male rats, ginger extract supplementation into the diet significantly reduced neuropathic pain-associated behaviors (hypersensitivity), emotional responses, and spontaneous pain) in neuropathic rats. Ginger extract supplementation also improved intestinal integrity of the colon and decreased neuroinflammation (NF-κB, TNF-α) in the colon and the brain (amygdala, a brain region involved in emotional-affective behavior and pain modulation). In female rats, ginger extract supplementation also decreased neuropathic pain-associated behaviors hypersensitivity. This study demonstrated that ginger extract supplementation mitigates neuropathic pain via mitigating intestinal permeability and neuroinflammation, pointing to an action in the gut-brain connection, predominantly in males. Ginger extract mitigated neuroinflammation and improved mitochondrial homeostasis in the dorsal root ganglion (DRG) and spinal cord (SC): In male and female rats, ginger extract supplementation increased tight junction protein (claudin-3) in the peripheral nervous system (DRG) and decreased neuroinflammation measured as decreased markers of astrocyte activation (GFAP, in DRG of males and females and SC of females), microglia activation (IBA1, in DRG of females and SC of males; CD11b in DRG and SC of males), immune response (TLR4 in DRG and SC of males and females; TLR2 in SC of males and DRG and SC of females), and proinflammatory transcription factor and cytokines (NFkB, TNFα, and IL1β). Ginger extract supplementation modulated mitochondrial biogenesis (increased TFAM in SC of males and DRG in females; decreased PGC1α in DRG and SC of males and DRG of females), fission (decreased FIS1 in DRG, increased FIS1 in SC of males, and increased FIS1 in DRG of females; decreased DRP1 in DRG of males and SC of males and females), fusion (MFN2 and MFN1 in DRG and SC of both sexes), and mitophagy (decreased PINK1 in DRG and SC of both sexes). Ginger extract supplementation significantly reduced the concentrations of gut-derived short-chain fatty acids (SCFA) (i.e., acetic acid, butanoic acid, hexanoic acid, propanoic acid, and valeric acid) and plasma branched-chain amino acids (BCAA). This study demonstrated that ginger extract supplementation reduced neuroinflammation, improved mitochondrial homeostasis, and decreased gut-derived SCFA concentrations and plasma BCAA in rat model of neuropathic pain. Correlations between effects of ginger extract on pain, gut microbiome composition, and neuroinflammation (in male NP rats) Ginger extract supplementation significantly reduced hypersensitivity in a neuropathic pain model in males. The ginger extract group had increases/decreases in 16 (10/6) genes and 11 (1/10) genes related to neuroinflammation, respectively. Specifically, ginger extract supplementation downregulated Fos and Gadd45g genes and upregulated Hdac2 genes in the brain (amygdala). Based on the gut microbiome composition results, ginger extract supplementation favored gut microbiome composition in the SNL male rats, as shown by a decreased abundance of Rikenella, Muribaculaceae, Clostridia UCG-014, Mucispirillum schaedleri, RF39, Acetatifactor, and Clostridia UCG-009, and an increased abundance of Flavonifactor, Hungatella, Anaerofustis stercorihominis, and Clostridium innocuum group. Less hypersensitivity was positively correlated with the expression of Cd300lf and the abundance of Ruminococcaceae_UBA1819 and Flavonifractor, and was negatively correlated with the abundance of UGC-010, Lachnospiraceae_FCS020_group, and Bacteroides massiliensis. This study demonstrated that ginger extract alleviated neuropathic pain by reversing neuroimmune signaling in the amygdala and by modulating the gut microbiome in male rats, providing further evidence for an action on the microbiota-gut-brain axis. Correlation between the effects of ginger on gut microbiome composition, fecal metabolites, and amygdala neuropathology genes (in female NP rats) Ginger extract supplementation increased the abundance of Bacteroidota phyla (i.e., Rikenella, Alistipes, Muribaculaceae, Odoribacter), Firmicutes (i.e., UBA1819, Ruminococcaceae, Oscillospiraceae, Roseburia), and Verrucomicrobiota (i.e., Victivallis) in female rats with neuropathic pain. Ginger extract supplementation increased the levels of nine hydrophilic positive metabolites (e.g., val-glu, urocanic acid, oxazolidinone, L-threonine, L-norleucine, indole, imino-tryptophan, 2,3-octadiene-5,7-diyn-1-ol, and (2E)-3-(3-hydroxyphenyl)acrylaldehyde), increased the levels of 2 hydrophilic negative metabolites (e.g., methylmalonic acid and metaphosphoric acid), and decreased the levels of 5 hydrophilic negative metabolites (e.g., xanthine, N-acetylmuramic acid, doxaprost, adenine, and 1-myristoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), suggesting beneficial effects on anti-neuroinflammation. Among the 770 neuropathology genes measured in the brain (amygdala), ginger extract supplementation caused the upregulation of 9 genes (APC, CCNH, EFNA5, GRN, HEXB, ITPR1, PCSK2, TAF9 and WFS1) and downregulation of 3 genes (AVP, C4A and TSPO). This study demonstrated that ginger extract supplementation mitigated neuropathic pain-associated behaviors in female rats, in part, by reversing the molecular neuropathology signature of the amygdala and by the modulation of the gut microbiome composition and fecal metabolites. The dosages of 200, 400, and 600 mg ginger extract/kg BW for rats correspond to 45 g, 90 g, and 135 g raw ginger for human daily consumption, respectively. In general, the limited ginger extract dose-response effects on pain behaviors, neuroinflammation, and mitochondrial homeostasis suggest that the 45 g dose of raw ginger could be used for human daily intake to achieve potential benefits in the management of neuropathic pain.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Santos JM, Deshmukh H, Elmassry MM, Yakhnitsa V, Ji G, Kiritoshi T, Presto P, Antenucci N, Liu X, Neugebauer V, Shen CL (corresponding author). Beneficial effects of ginger extract on pain behaviors, inflammation and mitochondrial function in the colon and different brain regions of male and female neuropathic rats: a gut-brain axis. Nutrients. 2024; 16(20): 3563. doi: 10.3390/nu16203563.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Shen CL, Deshmukh H, Santos JM, Elmassry MM, Presto P, Driver D, Bhakta V, Yakhnitsa V, Kiritoshi T, Ji G, Lovett J, Hamood A, Neugebauer V. Fecal microbiota transplantation modulates gut microbiome composition and glial signaling in brain and colon of rats with neuropathic pain: evidence for microbiota-gut-brain axis. Journal of Frailty & Aging, 2024. https://doi.org/10.14283/jfa.2024.65
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: 143. Appell C, Jiwan NC, Shen CL, Luk HY. Curcumin mitigates muscle atrophy potentially by attenuating calcium signaling and inflammation in a spinal nerve ligation model. Current Issues in Molecular Biology. 2024; 46(11), 12497 - 12511; https://doi.org/10.3390/cimb46110742
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: 142. Jiwan N, Appell C, Shen CL, Luk HY. The effect of geranylgeraniol and ginger on satellite cells myogenic state in type 2 diabetic rats. Current Issues in Molecular Biology. 2024; 46(11), 12299 - 12310; https://doi.org/10.3390/cimb46110730.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: 137. Shen CL, Santos JM, Elmassry MM, Bhakta V, Driver Z, Ji G, Yakhnitsa V, Kiritoshi T, Lovett J, Hamood AN, Sang S, Neugebauer V. Ginger polyphenols reverse molecular signature of amygdala neuroimmune signaling and modulate microbiome in male rats with neuropathic pain: Evidence for microbiota-gut-brain axis. Antioxidants, 2024, 13, 502. https://doi.org/10.3390/ antiox13050502
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: 136. Shen CL, Wang R, Santos JM, Elmassry MM, Stephens E, Neugebauer V. Ginger alleviates mechanical hypersensitivity and anxio-depressive behavior in rats with diabetic neuropathy through beneficial actions on gut microbiome composition, mitochondria and neuroimmune cells of colon and spinal cord. Nutrition Research. 2024; 124, 73 - 84. Doi: 10.1016/j.nutres.2024.01.014.

Progress 01/01/23 to 12/31/23

Outputs
Target Audience:During the period covered by this 3rd annual report, this project has provided research/experiential learning opportunities/laboratory instruction to 2 medical students and 3 undergraduate students through formal educational programs, namely (i) Year 2 Medical Student Research Program for medical students, and (ii) Undergraduate Research Scholar Program and Pi2 Program for undergraduate students. Two of the five trainees are racial and ethnic minorities. Changes/Problems:There were no major changes in approved protocols during the reporting period. However, the delays, including (i) confirmation of gene expression (mRNA) using western blot or IHC and (ii) confirmation of statistical analysis results and correlation analysis, will need more time to accomplish. What opportunities for training and professional development has the project provided?We provided training in animal behavior, surgery, animal handling, molecular biology, data analysis, interpretation and presentation to two senior research associates and the students for their professional development in the field of bioactive compounds in pain research. How have the results been disseminated to communities of interest?We published our work in 2 peer-referred journals in the area of bioactive compounds for neuropathic pain. Santos JM, Wang R, Bhakta V, Driver Z, Vadim Y, Kiritoshi T, Ji G, Neugebauer V, Shen CL (cooresponding author). Turmeric bioactive compounds ameliorate neuropathic pain by beneficial actions on glial activation, mitochondrial function, and oxidative stress in rat spinal cord and amygdala. Nutrients2023,15(20), 4403;https://doi.org/10.3390/nu15204403 Appell CR, Jiwan NC, Wang R, Shen CL, Luk HY. Ginger supplementation attenuated mitochondrial fusion and improved skeletal muscle size in type 2 diabetic rats. In Vivo (in press). In addition, we have four manuscripts are currently under review for journal publication. Shen CL, Wang R, Santos JM, Elmassry MM, Stephens E, Neugebauer V. Ginger alleviates mechanical hypersensitivity and anxious-depression-like behavior in rats with diabetic neuropathy by altering the gut microbiome and suppressing spinal microglia activation: microbiota-gut-CNS-axis. Submitted to Journal of Agricultural and Food Chemistry. Shen CL, Santos JM, Elmassry MM, Bhakta V, Driver Z, Ji G, Yakhnitsa V, Kiritoshi T, Lovett J, Hamood AN, Sang S, Neugebauer V. Ginger polyphenols mitigate hypersensitivity in neuropathic pain rats by reversing the molecular signature of amygdala neuroimmune signaling: implication of microbiome-gut-brain-axis. Submitted to Journal of Functional Foods. Jiwan NC, Appell CR, Sterling R, Shen CL, Luk H-Y. The Effect of geranylgeraniol and ginger on satellite cells myogenic state in diabetic rats. Submitted to Journal of Nutritional Biochemistry. Although we plan to perform more data analysis in the requested NCE grant period (Year 4), the results so far suggest that ginger has beneficial effects on pain behaviors through actions that involve decreased inflammatory signaling in the gut-brain-axis and improved gut health. Therefore, this research project will impact the emerging field of neuropathic pain management through natural food ingredients (ginger root and its bioactive compounds) affecting the gut-brain-axis. What do you plan to do during the next reporting period to accomplish the goals?We would like to request a one-year no-cost extension (Year 4). This is important so that we can (i) confirm the findings of gene expression (mRNA) in frontal cortex, spinal cord, and colon at the protein level, using western blot or IHC (SA 3), (ii) confirm statistical analysis results for fecal metabolites and neuroinflammation gene profiles in both male and female studies (SA 2), and (iii) perform the proposed correlation analysis (SA 4). We plan to submit 3-4 abstracts for presentation at national or international conferences. Finally, we aim to submit 3-4 manuscripts in high-impact journals to address GEG dose-response and sex differences in the outcome measures.

Impacts
What was accomplished under these goals? We have completed two animal studies to deliver gingerols-enriched ginger (GEG) via oral gavage in both male animals (Male Study) and female animals (Female Study). Male animal study There were 5 groups, i.e., sham+vehicle (corn oil) [Sham group], spinal nerve ligation (spinal nerve ligation, a neuropathic pain model)+vehicle (corn oil) [SNL group], SNL+200 mg GEG/BW [GEG200 group], SNL+400 mg GEG/BW [GEG400 group], and SNL+600 mg GEG/BW [GEG600 group] for 4-week periods. We performed laboratory work for the proposed SA described below. SA 1 included pain-associated behavioral outcomes (mechanical hypersensitivity, spontaneous pain, emotional pain, and anxiety). SA 2 focused on gut health (intestinal barrier integrity: plasma lipopolysaccharide binding protein, gene expression (mRNA) of tight junction protein; gut microbiome, and fecal non-targeted metabolites). SA 3 measured neuroinflammation gene profiles (RT-PCR confirmation in amygdala as well as gene expression of neuroinflammation- and mitochondrial function-associated genes in frontal cortex, amygdala, spinal cord, dorsal root ganglion, and colon). SA 4 correlation analysis. Besides statistical analysis for SA 2 (confirmation of fecal metabolites), SA 3 (confirmation of neuroinflammation gene profiles) and SA 4 (correlation analysis) is underway, we still need to conduct western blot or IHC to confirm the gene expression (mRNA) findings in collected tissues (frontal cortex, spinal cord, and colon). Female animal study Similar to the Male Study, there were 5 groups, i.e., sham+vehicle (corn oil) [Sham group], spinal nerve ligation (SNL, pain model)+vehicle (corn oil) [SNL group], SNL+200 mg GEG/BW [GEG200 group], SNL+400 mg GEG/BW [GEG400 group], and SNL+600 mg GEG/BW [GEG600 group] for 4-week periods] for the same SA 1-4. We performed laboratory work as proposed, except for statistical analysis for SA 2 (confirmation of fecal metabolites), SA 3 (confirmation of neuroinflammation gene profiles) and SA 4 (correlation analysis) as well as protein confirmation using western blot or IHC in collected tissues (frontal cortex, spinal cord, and colon). We would like to request for a one-year no-cost extension (Year 4). In this upcoming 4th year, we plan to (i) confirm the findings of gene expression (mRNA) in frontal cortex, spinal cord, and colon, using western blot or IHC (SA 3), (ii) perform statistical analysis for SA 2 (confirmation of fecal metabolites), SA 3 (confirmation of neuroinflammation gene profiles) and SA 4 (correlation analysis) in both male and female studies, (iii) submit 3-4 abstracts for presentation at national or international conferences, and (iii) publish 3-4 manuscripts in high-impact journals. Findings of GEG supplementation at the chronic stage of the SNL model: (1) Spontaneous pain behavior in female animals. Quantification of spontaneous (i.e., tonic, non-evoked) pain was done with the Rodent Grimace Test (RGT). After 4 weeks of GEG intervention, compared to the SNL vehicle group, the SNL female animals with GEG supplementation showed significantly decreased spontaneous pain behavior, reflected in lower scores of RGT parameters, i.e., orbital tightening, nose bulge, ear position, and whiskers changes. A limited GEG-dose response was observed. (2) Gene expression (mRNA) in tissues collected from female animals. The effects of GEG supplementation were observed in all GEG-treated SNL groups (p<0.05); specifics and exceptions are noted below. gene expression (mRNA) levels were determined in frontal cortex (FC), amygdala (AMY), spinal cord (SC), dorsal root ganglion (DRG), and colon. Tight junction (claudin-3). GEG increased the claudin-3 gene expression in AMY and DRG, while GEG had no effect on claudin-3 gene expression in FC, SC, and colon. Pro-inflammatory cytokines (NF-kB, TNF-α and IL-1β) gene expression. GEG suppressed NF-kB gene expression in FC (GEG400), AMY, SC, DRG, and colon (GEG400). GEG suppressed TNF-α gene expression in FC, AMY (GEG600), DRG, and colon; while GEG had no effect on TNF-α gene expression in SC. GEG suppressed IL-1β gene expression in FC, AMY, SC (GEG200), DRG, and colon (GEG400, GEG600). Glial activation (IBA-1, GFAP, and CD11B). GEG decreased IBA-1 gene expression in FC (GEG200, GEG400), DRG, and colon (GEG400, GEG600, 0.05
Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2023 Citation: Santos JM, Wang R, Bhakta V, Driver Z, Vadim Y, Kiritoshi T, Ji G, Neugebauer V, Shen CL. Turmeric bioactive compounds ameliorate neuropathic pain by beneficial actions on glial activation, mitochondrial function, and oxidative stress in rat spinal cord and amygdala. Nutrients 2023, 15(20), 4403; https://doi.org/10.3390/nu15204403

Progress 01/01/22 to 12/31/22

Outputs
Target Audience:During the period covered by this 2nd annual report, this project has provided research/experiential learning opportunities/laboratory instruction to 2 medical students and 3 undergraduate students through formal educational programs, namely (i) Year 2 Medical Student Research for medical students, and (ii) Undergraduate Research Scholar Program and Pi2 Program for undergraduate students. Two of the five trainees are racial and ethnic minorities. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Through animal studies, we provided training in behavior, molecular biology, surgery, animal handling, data analysis, interpretation and presentation to two senior research associates for their professional development in the field of bioactive compounds in pain research. How have the results been disseminated to communities of interest?We published our work in 3 peer-referred journals. Paper #1: Shen et al. Dietary supplementation of gingerols- and shogaols-enriched ginger root extract attenuate pain-associated behaviors while modulating gut microbiota and metabolites in rats with spinal nerve ligation. Journal of Nutritional Biochemistry. 2022 Feb;100:108904. doi: 10.1016/j.jnutbio.2021.108904. Paper #2: Shen CL et al. Gingerol-enriched ginger supplementation mitigates neuropathic pain via mitigating intestinal permeability and neuroinflammation: gut-brain connection. Frontiers in Pharmacology. 2022. Jul 8;13:912609. doi: 10.3389/fphar.2022.912609. Paper #3: Wang/Shen (corresponding author). Ginger root extract improves GI health in diabetic rats by improving intestinal integrity and mitochondrial dysfunction. Nutrients. 2022 Oct 19;14(20):4384. doi: 10.3390/nu14204384. In addition, we published two invited review articles. Review #1: Shen et al. Bioactive compounds for neuropathic pain: an update on preclinical studies and future perspectives. Journal of Nutritional Biochemistry. 2022; 104:108979. doi: 10.1016/j.jnutbio.2022.108979. Review #2: Shen et al. Bioactive compounds for fibromyalgia-like symptoms: a narrative review and future perspectives. International Journal of Environment Research and Public Health, Special Issue: "Individual Difference in Pain in Various Populations". 2022, 19(7):4148. Although we are still generating more data through upcoming animal studies in 3rd year grant period, the results so far suggest that this research project will impact the emerging field of neuropathic pain management through natural food ingredients (ginger root and its bioactive compounds) affecting the gut-brain-axis. What do you plan to do during the next reporting period to accomplish the goals?We plan to finish the bench work and data analysis for non-targeted fecal metabolites in both male and female studies. We plan to finish bench work and data analysis for all mRNA expression in collected tissues (amygdala, frontal cortex, spinal cord, DRG, and colon) of female study. We will conduct immunohistochemistry (IHC) or Western blotting to confirm the findings of mRNA expression at the protein level in colon or other available tissues of both male and female studies. We plan to submit 3-4 abstracts for presentation at national or international conferences. Finally, we aim to publish 2-3 manuscripts to address GEG dose-response and sex differences in the outcome measures.

Impacts
What was accomplished under these goals? We have conducted two animal studies to deliver gingerols-enriched ginger (GEG) via oral gavage inanimals with neuropathic pain. Male animal study There were 5 groups including sham+vehicle (corn oil), spinal nerve ligation (spinal nerve ligation, SNL, neuropathic pain model)+vehicle (corn oil), SNL+200 mg GEG/BW, SNL+400 mg GEG/BW, and SNL+600 mg GEG/BW for 4-week periods. We assessed behavioral outcomes (SA 1) and collected tissues for mRNA expression of neuroinflammation (SA 3) in frontal cortex, amygdala, spinal cord, dorsal root ganglion (DRG), and colon. The cecum feces were collected for gut microbiome and non-targeted metabolites (hydrophobic and hydrophilic) analysis. All behavioral outcomes (SA 1), assessed by von Frey test, vocalization measurements, open field test (OFT), elevated plus maze test (EPM), and grimace test were completed with statistical analysis. In terms of gut health (SA 2), (1) statistical analysis of gut microbiome data is completed; (2) the laboratory work on fecal metabolites is currently being conducted at TTU core facility; (3) intestinal barrier integrity was assessed by completion of mRNA expression analysis of tight junction protein (i.e., claudin-1, claudin-3, occuldin, and zonulin) in variety of tissues and urinary lactulose/mannitol concentrations; and (4) intestinal inflammation was assessed by completion of mRNA expression analysis of TNF-α, NF-kB, and IL-1β in colon. Regarding NP-associated neuroinflammation (SA 3), we have completed the mRNA gene expression analysis of glial activation biomarkers, mitochondrial function parameters (fission, fusion, biogenesis, respiratory chain complex, and oxidative stress), and proinflammatory cytokine/chemokine signaling in all collected tissues. Female animal study Similar to the Male Study, there were 5 groups [sham+vehicle (corn oil), spinal nerve ligation (SNL, pain model)+vehicle (corn oil), SNL+200 mg GEG/BW, SNL+400 mg GEG/BW, and SNL+600 mg GEG/BW for 4-week periods] in the Female Study. We have completed animal feeding experiment, data collection of pain-associated behavior outcomes, collection of specimens (feces, tissues), and data analysis of gut microbiome. The remaining laboratory work (non-target fecal metabolites, mRNA expression, IHC) and follow-up statistical analyses will be conducted/completed in the Year 3. Effect of GEG supplementation: (1) Pain hypersensitivity. Pain mechanosensitivity was assessed by von Frey test and by paw compression test (reflex thresholds). Compared to the SNL vehicle group, after 4 weeks of GEG intervention, all GEG doses (200, 400, and 600 mg/kg body weight) significantly mitigated SNL-induced mechanical hypersensitivity regardless of sex. (2) Emotional pain responses. Emotional pain responses were assessed by measuring audible and ultrasound vocalization evoked by innocuous or noxious stimuli. In the male study, the magnitude of decrease of SNL-induced emotional responses was greater in the 200 mg/BW GEG group than in the other GEG groups (400 and 600 mg/kg BW). A similar trend in GEG effects on emotional pain responses was observed in the female animals with NP. (3) Spontaneous pain behavior. Quantification of spontaneous pain was done with the Rodent Grimace Test (RGT). After 4 weeks of GEG intervention, compared to the SNL vehicle group, the SNL male animals with GEG supplementation showed significantly decreased spontaneous pain behavior, reflected in lower scores of orbital tightening, nose budge, ears position, and whiskers changes, in a GEG-dose dependent manner. (4) Pain-associated anxiety. Pain-associated anxiety behaviors were assessed by OFT and EPM. After 4 weeks of GEG intervention, in both male and female, the low dose of GEG (200 mg/kg BW) decreased pain-associated anxiety behaviors, as shown by increased duration/frequency in the center of OFT and by increased distance travelled and duration in the open-arms of EPM. (5) Gut microbiom. Based on alpha-diversity analysis (Pielou's evenness and Faith's phylogenetic diversity indices), we observed a statistically significant drop in species evenness and richness in groups treated with GEG of both male and female animals with NP (Wilcoxon signed-rank test, P < 0.05). SNL induction resulted in the loss of some of the microbiome species. These taxa belonged to several phyla, such as Proteobacteria (e.g., Sutterella stercoricanis) and Firmicutes. This effect of GEG was more pronounced in female rats, than in male rats. To answer Q2, we used a similar approach as for Q1 and examined the increase/decrease of species in GEG groups in both sexes vs. SNL using LOCOM. First, it is remarkable that certain species are consistently altered by GEG treatments in both sexes. This was the case for ASVs of Rikenella, Muribaculaceae, Clostridia UCG-014, and Mucispirillum schaedleri, RF39, which were decreased in GEG treated animals (both sexes) vs. SNL. In contrast, ASVs of the following taxa were increased in GEG treated animals (both sexes) vs. SNL: Bacteroides, UBA1819, and Hungatella. Second, we observed distinct patterns between sexes and their response to GEG treatments. For example, an Anaerotruncus ASV was only decreased in male rats. Flavonifractor and Anaerofustis stercorihominis were only increased in male rats. Parasutterella and Odoribacter were decreased in female rats only, while Victivallis was increased. Third, a few taxa showed a dose-response with GEG treatments, such as RF39 and UCG-009, Parasutterella, Hungatella, and Rikenella. (6) mRNA in in brain, spinal cord, DRG, and colon. Tight junction: claudin-3 in collected tissues. GEG supplementation increased the mRNA expression of claudin-3 in the amygdala, DRG, colon, but not in frontal cortex and spinal cord. Pro-inflammatory cytokines: NF-kB, TNF-α, IL-1β, and IL-6. GEG supplementation suppressed SNL-induced gene expression of NF-kB, TNF-α, and IL-1β in the above tissues. Glial activation: IBA-1, CD11B, and GFAP. GEG supplementation at high doses (400 and 600 mg/kg BW GEG) significantly increased gene expression of IBA-1 (frontal cortex and spinal cord) and GFAP (frontal cortex), but it significantly decreased gene expression of CD11b (frontal cortex, amygdala, spinal cord, DRG, and colon), IBA-1 (amygdala and colon), CD11b (), and GFAP (amygdala, DRG, and colon). Mitochondrial fission: DRP1 and FIS1. GEG supplementation significantly increased mRNA expression of DRP1 in frontal cortex and amygdala, while it significantly decreased DRP1 mRNA expression in colon. In terms of FIS1, SNL induction significantly decreased the mRNA expression levels of FIS1 in colon, while GEG at 200 mg/kg BW reversed such this decrease. Mitochondrial fusion: MFN1 and MFN2. GEG supplementation significantly decreased MFN1 (amygdala, spinal cord, DRG, and colon) and MFN2 (amygdala, spinal cord, DRG, and colon), but significantly increased the mRNA expression MFN1 in frontal cortex. Mitochondrial biogenesis: PINK1, PGC-1α, NRF1, and TFAM. GEG supplementation significantly suppressed SNL-induced mRNA expression of PINK1 in frontal cortex, spinal cord, DRG, and colon. Although SNL did not affect PINK1 mRNA expression in amygdala, GEG supplementation significantly increased PINK1 mRNA expression level in amygdala. GEG supplementation significantly increased SNL-suppressed mRNA expression of PGC1a in all collected tissues (frontal cortex, amygdala, spinal cord, DRG, and colon). GEG supplementation significantly increased mRNA expression of NRF1 (frontal cortex, amygdala, and spinal cord) and TFAM (frontal cortex and spinal cord), while it significantly reduced SNL-induced NRF1 mRNA expression in colon. Mitochondrial respiratory chain complexes: complex I and complex III. GTP supplementation significantly increased mRNA expression of complex I (fontal cortex, amygdala, spinal cord, DRG, and colon) and complex III (frontal cortex, amygdala, spinal cord, DRG, and colon).

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: " Shen CL, Wang R, Ji G, Elmassry MM, Zabet-Moghaddam M, Vellers H, Hamood AN, Gong X, Mirzaei P, Sang S, Neugebauer V. Dietary supplementation of gingerols- and shogaols-enriched ginger root extract attenuate pain-associated behaviors while modulating gut microbiota and metabolites in rats with spinal nerve ligation. Journal of Nutrition Biochemistry. 2022 Feb;100:108904. doi: 10.1016/j.jnutbio.2021.108904.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: " Wang R, Santos JM, Dufour JM, Stephens ER, Miranda JM, Washburn RL, Hibler T, Kaur G, Lin D, Shen CL (corresponding author). Ginger root extract improves GI health in diabetic rats by improving intestinal integrity and mitochondrial dysfunction. Nutrients. 2022 Oct 19;14(20):4384. doi: 10.3390/nu14204384.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: " Shen CL, Wang R, Yakhnitsa V, Santos J, Watson C, Kiritoshi T, Ji C, Hamood A, Neugebauer V. Gingerol-enriched ginger supplementation mitigates neuropathic pain via mitigating intestinal permeability and neuroinflammation: gut-brain connection. Frontiers in Pharmacology. 2022. Jul 8;13:912609. doi: 10.3389/fphar.2022.912609. eCollection 2022.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: " Shen CL, Castro L, Fang C-Y, Castro M, Sherali S, White S, Wang R, Neugebauer V. Bioactive compounds for neuropathic pain: an update on preclinical studies and future perspectives. Journal of Nutritional Biochemistry. 2022; 104:108979. doi: 10.1016/j.jnutbio.2022.108979. Online ahead of print.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: " Shen CL, Schuck A, Tompkins C, Dunn DM, Neugebauer V. Bioactive compounds for fibromyalgia-like symptoms: a narrative review and future perspectives. International Journal of Environment Research and Public Health, Special Issue: Individual Difference in Pain in Various Populations. 2022, 19(7):4148.

Progress 01/01/21 to 12/31/21

Outputs
Target Audience:This project has provided research/experiential learning opportunities/laboratory instruction to 2 medical students and 3 undergraduate students through formal educational programs, namely (i) Medical Student Summer Research Program and Year 2 Medical Student Research for medical students, and (ii) Undergraduate Research Scholar Program and Pi2 Program for undergraduate students. Two of the five trainees are racial and ethnic minorities. Changes/Problems:The major changes of this project include: A change in the method of GEG delivery from dietary supplementation to oral gavage, which has been approved by TTUHSC IACUC full board meeting. Based on our pilot study, we learned that oral gavage avoids the GEG taste issue. Replace lipopolysaccharide binding protein (LBP) in plasma samples test with urinary lactulose and mannitol ratio test for intestinal permeability assessment (SA 3). We tried to quantify the levels of lipopolysaccharide binding protein (LBP) in plasma samples to access the intestinal barrier integrity. However, the concentrations of LBP in plasma was below the detectable limits, suggesting the LBP method may not be sensitive enough to detect such subtle change due to SNL or GEG supplementation. This change has been approved by TTUHSC IACUC full board meeting. Replace Conditioned Place Preference (CPP)/Conditioned Place Aversion (CPA) tests that require animal training/learning with Grimace test for ongoing pain-associated behavioral outcomes in SA 1. This change has been approved by TTUHSC IACUC full board meeting. Ongoing pain will be assessed as pain scores in freely moving rats using facial grimace chart (Sotocinal et al 2011), which has been used successfully in neuropathic pain models (Akintola et al 2017).Method:Rats are acclimated to the testing room for 1 hour before the beginning of the experiment. Animals are placed in individual Plexiglas cages with bedding from animal's home cage to make the environment as stress-free as possible. Animals are acclimated to the cage for 5 to 10 minutes before pictures are taken with a digital camera (4 pictures at 20-second intervals to increase reliability). If the animal was grooming, actively sniffing or rearing, then the next available picture is used for scoring. Images are taken from multiple angles. For consistency, the same individuals score all the images in a blinded manner. Five facial parameters (action units) are scored: orbital tightening (eye lid fissure), nose bulge, cheek bulge, ear position, and whisker change. Each action unit is scored 0 (not present), 1 (moderately visible), or 2 (severe) on the basis of criteria described previously (Sotocinal et al 2011). An initial grimace score of each photograph is calculated by averaging the scores of the 5 action units, and a mean grimace score is obtained from the 4 images. Scores from the blinded investigators are averaged for the final result. All above changes will have no significant impact on either the overall goal of the project or the rate of expenditure. Instead, Grimace test will provide a less invasive/stressful approach for the involved animals, than CPP/CPA test. References: Akintola T, Raver C, Studlack P, Uddin O, Masri R, Keller A. The grimace scale reliably assesses chronic pain in a rodent model of trigeminal neuropathic pain.Neurobiol Pain. 2017 Aug;2:13-17. PMID: 29450305 Sotocinal, S.G., Sorge, R.E., Zaloum, A., Tuttle, A.H., Martin, L.J., Wieskopf, J.S.,Mapplebeck, J.C., Wei, P., Zhan, S., Zhang, S., McDougall, J.J., King, O.D., Mogil,J.S., 2011. The Rat Grimace Scale: a partially automated method for quantifying painin the laboratory rat via facial expressions. Mol. Pain 755. What opportunities for training and professional development has the project provided?Through animal studies, we provided training to two senior research associates for their professional development in the field of bioactive compounds in pain research. How have the results been disseminated to communities of interest?We published our pilot study (Shen et al. Dietary supplementation of gingerols- and shogaols-enriched ginger root extract attenuate pain-associated behaviors while modulating gut microbiota and metabolites in rats with spinal nerve ligation) in the Journal of Nutritional Biochemistry (JNB), special issue: precision nutrition-individual nutrient needs for optimal health throughout the lifespan this year (doi: 10.1016/j.jnutbio.2021.108904). JNB has impact factor of 6.048 and open access to all communities of interest. In addition, one review manuscript (Shen et al. Bioactive compounds for neuropathic pain: an update on preclinical studies and future perspectives) is currently under review by the JNB. Although we are still generating more data through upcoming animal studies in our 2nd and 3rd year grant periods, the results so far suggest that this research project will impact the emerging field of neuropathic pain management through natural food ingredients (ginger root and its bioactive compounds) affecting the gut-brain-axis. What do you plan to do during the next reporting period to accomplish the goals?We plan to conduct two animal studies; one animal study on male rats and another on female rats. A total of 50 (male SD rats) will be divided into 5 groups (n=10 per group): Sham+vehicle, SNL+vehicle, SNL+200 mg/kg GEG, SNL+400 mg/kg GEG, and SNL+600 mg/kg GEG for 4 weeks. We will perform sensory and affective behaviors (SA 1) while animals are alive. Collected tissues from animals will be used to perform SA 2: gut health; SA 3: neuroinflammation via mRNA expression, protein expression, ELSIA, or IHC, in the amygdala (right), spinal cord (lumbar enlargement), dorsal root ganglion (DRG), and GI. SA 4: gut microbiota composition and fecal metabolites). In SA 3, we will use the following approaches: (1) due to the limited amount of tissue (e.g. amygdala-right, DRG), mRNA gene expression using RT-PCR will be our first priority; and (2) if adequate tissue samples are available, we will perform IHC and/or protein expression to confirm the findings of mRNA expression. We will analyze the amygdala (right-side, because of hemispheric lateralization of pain processing), spinal cord (lumbar enlargement, site of afferent input from nerve injury), and DRG (L5+L6, site of afferent input from nerve injury).

Impacts
What was accomplished under these goals? We conducted the following two animal studies: STUDY 1 Gingerol-enriched ginger root extract (GEG) was delivered to male SD rats via diet. The groups assignment included wild type control, sham, spinal nerve ligation (SNL, pain model), SNL+0.375%GEG, SNL+0.75%GEG, and SNL+1.125%GEG for 4-week feeding periods. Unexpectedly, GEG administration through the diet turned out to be problematic because the animals did not like the taste of GEG; we lost all animals (n=6) from the SNL+1.125% GEG group, 1 animal from the SNL+0.75%GEG group, and 1 animal from the SNL+0.375%GEG group within a week after SNL. Despite that, we still completed the 4-week intervention period, performed behavioral assessment (SA 1), conducted intestinal permeability test (SA 3), and collected tissues for mRNA expression of neuroinflammation (SA 3) in colon, amygdala (right and left), spinal cord (lumbar enlargement), and ileum. Preliminary findings: (1) GEG supplementation mitigated pain hypersensitivity. Pain mechanosensitivity was assessed by von Frey test and by tissue compression (spinal reflexes). According to the results of von Frey test, compared to the SNL group, both SNL+0.375% GEG and SNL+0.75% GEG groups showed reduced pain sensitivity as early as 1-week post-operation and sustained through 4 weeks, as shown by increased mechanical thresholds. At the end of the study (4 weeks after supplements started), the order of pain sensitivity was SNL group > SNL+0.75%GEG group > SNL+0.37%%GEG group > sham group = control group. The findings of spinal reflexes were similar to those in von Frey test. (2) GEG supplementation decreased NP-induced audible vocalizations. At the baseline, there were no differences in audible vocalization among all groups, regardless of different stimuli conditions, including innocuous mechanical stimuli, innocuous ultrasound stimuli, noxious mechanical stimuli, and noxious ultrasound stimuli. GEG supplementation significantly reduced SNL-induced emotional responses under all statuses. (3) GEG supplementation decreased NP-induced spontaneous pain. Quantification of spontaneous pain was assessed by Grimace test. At the baseline, there were no any differences in any facial expressions. At the end of study, GEG supplementation significantly reduced SNL-induced nose bulge, whiskers change, and ear position of SNL-operated rats. (4) GEG supplementation tended to improve intestinal integrity. We evaluated intestinal permeability by analyzing urinary lactulose and mannitol levels. SNL procedure increased intestinal permeability as shown in increased ratio of lactulose/mannitol ratio. GEG supplementation had a trend in lowering the ratio of lactulose/mannitol, an indicator of intestinal permeability. (5) The expression levels of tight junction, mitochondrial, and inflammation genes in CNS (amygdala), PNS (spinal cord), and GI (colon only, not ileum) were modified by GEG supplementation. SNL procedure increased the expression levels of neuroinflammation-related genes. Amygdala: SNL operation tended to increase mRNA expression of zonulin (tight junction) (0.05 SNL+400 group > SNL+200 group > SNL group.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Shen CL, Wang R, Ji G, Elmassry MM, Zabet-Moghaddam M, Vellers H, Hamood AN, Gong X, Mirzaei P, Sang S, Neugebauer V. Dietary supplementation of gingerols- and shogaols-enriched ginger root extract attenuate pain-associated behaviors while modulating gut microbiota and metabolites in rats with spinal nerve ligation. Journal of Nutritional Biochemistry, Special Issues: Precision nutrition  individual nutrient needs for optimal health throughout the lifespan. 2021 Nov 5:108904. doi: 10.1016/j.jnutbio.2021.108904. JNB IF: 6.048, open access.
• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Shen CL, Castro L, Fang C-Y, Castro M, Sherali S, White S, Wang R, Neugebauer V. Bioactive compounds for neuropathic pain: an update on preclinical studies and future perspectives. Submitted to Journal of Nutritional Biochemistry on 9/23/2021, under review. Received favorable comments from reviewers. Plan to submit revision in the end of December 2021.