Articles in PresS. Physiol Genomics (August 18, 2017). doi:10.1152/physiolgenomics.00078.2017
Title: Chronic spinal cord changes in a high-fat diet fed male rat model of thoracic spinal contusion Running Title: Spinal cord injury and obesity
Authors: Redin A. Spann1, William J. Lawson1, Raymond J. Grill1 and Michael R. Garrett2 and Bernadette E. Grayson1
Affiliations: 1Department
of Neurobiology and Anatomical Sciences
2Department
of Pharmacology and Nephrology
University of Mississippi Medical Center, Jackson, MS 39216 Institution: University of Mississippi Medical Center, Jackson, MS Corresponding Author Bernadette E. Grayson University of Mississippi Medical Center Department of Neurobiology and Anatomical Sciences 2500 North State Street Jackson, MS 39216 Email: [email protected] Phone: 601-984-6809 Fax: 601-984-1655 Keywords: spinal cord injury, obese, high-fat diet, microarray ACKNOWLEDGEMENTS B.E.G is supported by the Department of Defense Award Number SC150016 and partially by the National Institute of General Medical Sciences Award Number P20GM104357. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health The work performed by M.R.G. through the UMMC Molecular and Genomics Facility is supported, in part, by funds from the National Institute of General Medical Sciences of the National Institutes of Health, including Mississippi INBRE (P20GM103476), Center for Psychiatric Neuroscience (CPN)-COBRE (P30GM103328) and Obesity, Cardio-renal and Metabolic Diseases-COBRE (P20GM104357).
1
Copyright © 2017 by the American Physiological Society.
ABBREVIATIONS ABCA1 ADH1 APOC4 ASGR1 CD68 DCKL2 GLP1R GPNMB HFD HMCGR HMOX1 HT2b IGF1 IGF2 LCAT MMP8 MMP12 PTGS1 tSCI TIMP1
ATP binding cassette subfamily A member 1 alcohol dehydrogenase 1 apolipoprotein C-IV asialoglycoprotein receptor 1 cluster of differentiation 68 doublecortin-like kinase 2, glucagon like peptide 1 receptor glycoprotein nmb high fat diet 3-hydroxy-3-methylglutaryl-CoA reductase heme oxygenase 1 serotonin receptor 2B insulin-like growth factor 1 insulin-like growth factor 2 lecithin cholesterol acyltransferase matrix metalloproteinase 8 matrix metalloproteinase 12 prostaglandin-endoperoxide synthase 1 thoracic spinal cord injury metallopeptidase inhibitor 1
2
ABSTRACT Individuals that suffer injury to the spinal cord can result in long-term, debilitating sequelae.
Spinal cord injured patients have increased risk for the development of
metabolic disease which can further hinder the effectiveness of treatments to rehabilitate the cord and improve quality of life. In the present study, we sought to understand the impact of high-fat diet induced obesity on spinal cord injury (SCI) by examining transcriptome changes in the area of the injury and rostral and caudal to site of damage 12 weeks after injury. Adult, male Long Evans rats received either thoracic level contusion of the spinal cord or sham laminectomy and then were allowed to recover on normal rat chow for 4 weeks and further on HFD for an additional 8 weeks. Spinal cord tissues harvested from the rats were processed for Affymetrix microarray and further transcriptomic analysis. Diverse changes in gene expression were identified in the injured cord in genes such as MMP12, APOC4, GPNMB and IGF1 and 2. The greatest signaling changes occurred in pathways involved in cholesterol biosynthesis and immune cell trafficking. Taken together, the cord changes in the chronically obese rat following thoracic spinal cord injury (SCI) injury reveal further potential targets for therapy. These could be further explored as they overlap with genes involved in metabolic disease.
3
INTRODUCTION To date, 275,000 Americans live with spinal cord injuries, with ~12,000 new cases per year (7). Approximately 45% of the injuries result in either complete or incomplete paraplegia from lesions of the thoracic, lumbar or sacral regions. Most impactful is that no effective treatments exist to permanently repair the injured spinal cord. Given the serious consequences of an injured spinal cord on every aspect of life, recovery of function and improvement of quality of life are imperative. Metabolic Syndrome (MetS) is a significant long-term problem for chronic spinal cord injury (SCI) patients (28, 30). MetS is a compilation of diseases which includes obesity, type-2 diabetes, dyslipidemia and cardiovascular disease (4). MetS may be exacerbated in the SCI population for a variety of reasons, including reduced or complete loss of physical activity (33). SCI individuals may be accelerated towards MetS due to change in body composition and physical activity coupled with altered calorie consumption (25, 35). However, when physical activity is statistically accounted for, specific components of MetS remain significant comorbidities of SCI (28, 30). SCI produces long-term motor, sensory and autonomic peripheral nervous system (PNS) dysregulation that may potentiate and exacerbate MetS in this population. Presently, over 2/3rds of the American population is overweight and about 1/3rd of the population is obese (10). Obesity is a significant component of MetS and in general is driven by modifiable life style factors which include consumption of calorie-dense, diets laden with saturated fats and refined carbohydrates (western type diet), reduced physical activity and exposure to chronic stress, among other risk factors. Obesity is a chronic lowgrade inflammatory disease characterized by an expansion of adipose tissue resulting in overall increased body weight (27). Due to disruption of the immune system as well as 4
for neurological and physiological reasons, obesity is associated with increased risk for diseases ranging from coronary artery disease to influenza, and complications during surgical procedures (13, 18, 24). Independently both SCI and MetS have serious long term ramifications. But taken together, a spinal cord injury coupled with MetS may negatively impact the trajectory of the SCI disease processes because of overlapping and integrated signaling systems common to both. Transcriptional changes after spinal cord injury have been extensively studied in lean animals (21, 31, 37), therefore, in the present study, we used a clinically-relevant rat model of spinal contusion injury coupled with diet-induced obesity to determine gene expression changes to the cord. Specifically, after either a thoracic spinal cord injury (tSCI) or sham laminectomy (sham), animals were allowed to recover on regular chow diet for 4 weeks and then provided a high-fat diet for an additional 8 weeks sufficient to produce obesity. Upon euthanasia, spinal cords were excised and microdissected. Cervical, thoracic and lumber level samples were processed for RNA. Affymetrix Microarray coupled with Genesifter© and Ingenuity Pathway Analysis© exploration of the data was performed on the thoracic compartment containing the lesion site with validation using samples from the three segments. A high degree of gene expression changes in cholesterol, inflammation and matrix protein pathways was observed. Additionally, it was also observed that trauma to the thoracic region of the spinal cord caused an altered gene profile in the uninjured cervical and lumbar regions. Taken together, thoracic level injury coupled with high-fat diet consumption causes significant durable transcriptome changes. METHODS
5
Animals: All procedures for animal use complied with the Guidelines for the Care and Use of Laboratory Animals by the National Institutes of Health and were reviewed and approved by the University of Mississippi Medical Center Institutional Animal Care and Use Committee. Male, Long Evans rats (400g) (Harlan, Indianapolis, IN) (N=10) were initially multiply housed and maintained in a room on a 12/12-h light/dark cycle at 25 °C and 5060% humidity with ad libitum access to water. Animals were maintained on standard chow (#8640, Envigo, 3.0 kCal/g; 17% fat, 54% carbohydrate, 29% protein).
Rats were
assigned to either sham-laminectomy (Sham) (N=5) or thoracic spinal cord injury (tSCI) (N=5) group in a counterbalanced fashion based on body weight on the day prior to the start of surgery. Surgery was performed and animals were allowed to recover for 4 weeks following surgery. Rats were singly housed from the time of surgery to the end of the study. Rats were switched to a palatable, high-fat diet (HFD) (#D03082706, Research Diets, New Brunswick, NJ, 4.54 kCal/g; 40% fat, 46% carbohydrate, 15% protein) for the remainder of the study totaling 8 weeks. Surgical procedures: All surgical procedures were performed on animals that were deeply anesthetized with an anesthetic cocktail (ketamine [80 mg/kg], xylazine [10 mg/kg], acepromazine [0.75 mg/kg]). tSCI surgeries were performed as previously described (29). Incisions were made on the animals’ dorsal skin and overlying muscles and the vertebral column was exposed. A laminectomy was performed at thoracic level 10 (T10) and the vertebral column was stabilized using forceps that grasp the ventral surface of the lateral spinous processes at vertebral levels T9 and T11. Using an Infinite Horizon Spinal Impactor Device (Precision Systems and Instrumentation, LLC, Fairfax
6
Station, VA), moderate contusion/compression injuries were delivered to the T10 spinal cord using 150 kdynes of force with a 1 sec dwell. The dura mater remained closed for the entire duration of SCI surgeries. Immediately following tSCI, the overlying muscles were sutured and the skin was securely closed using stainless steel wound clips. Sham surgeries were performed consisting of incisions to the animals’ dorsal skin exposing the musculature and vertebral column. A laminectomy was performed at thoracic level 10 (T10) and then the overlying muscles were sutured and the skin was securely closed using stainless steel wound clips. Postoperative animal care: Sham and tSCI rats received the following post-operative care with administration of: 1) an antibiotic (2.5mg/kg Baytril) once daily for a period of 10 days, 2) buprenorphine for post-surgical pain management (0.025 mg/kg, twice daily for a period of 5 days, then as needed); and 3) 3-5 mL of 0.9% saline, twice daily for a period of 3 days to ensure hydration. Beginning the day of tSCI surgery, each rat’s urinary bladder was manually expressed two to three times daily until the animal recovered the ability to void its bladder. Thoracic contusion results in disruption of the supraspinal pathways that are responsible for bladder voiding. In our hands, control of neurogenic bladder function returns in about 14 days. As a rule, bladder care was discontinued for an individual animal when it exhibited an already-voided bladder on two consecutive bladder care sessions. Body weight, composition: Following surgery, animals were weighed daily for the first 7 days and then weekly thereafter. Body lean and fat mass composition was analyzed using Echo Magnetic Resonance Imaging (echoMRI) (EchoMedical Systems, Houston,
7
TX) at 0 (time of surgery), 4 (start of HFD) and 11 wks (one week preceding the end of the experiment/euthanasia) . Tissue harvest: Twelve weeks following surgery, rats were euthanized by conscious decapitation starting at 6 h following the onset of the light cycle. The spinal cord was carefully excised using a rongeur instrument. The spinal cord was sectioned into three pieces measuring approximately 1.5 cm around each of the cervical, thoracic and lumbar enlargements. The T10 lesion site was completely included in the thoracic compartment. Tissue was flash frozen with methylbutane on dry ice and then stored in -80 ºC until further processing. Microarray analysis: Whole genome transcript analysis was performed using Affymetrix platforms from thoracic region spinal segments Sham and tSCI rats (n=4 per group). RNA was extracted using a QIAGEN miniprep RNA kit (QIAGEN, Inc, Valencia, CA) and evaluated for quality and integrity (Bio-Rad Experion™ System). All RNA gave RQI between 9-10. Spinal cord RNA were processed using manufacturer directions forGeneChip® 2.0 ST array and scanned using Affymetrix equipment (Scanner 3000 7G System). Hybridized chips were automatically washed, stained and scanned at the UMMC Institutional Molecular and Genomics Core using Affymetrix equipment. Data obtained from these gene expression studies are deposited in the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) with the GEO accession number XXXXXXX. Analysis of microarray data was performed using software provided by Affymetrix (Affymetrix® Expression Console™ Software) and commercially available GeneSifter™ software platform (http://www.genesifter.net). In brief, differentially expressed genes
8
evaluated by t-test using two methods: (1) FWER (family-wise error rate) procedure, p
Title: Chronic spinal cord changes in a high-fat diet fed male rat model of thoracic spinal contusion Running Title: Spinal cord injury and obesity
Authors: Redin A. Spann1, William J. Lawson1, Raymond J. Grill1 and Michael R. Garrett2 and Bernadette E. Grayson1
Affiliations: 1Department
of Neurobiology and Anatomical Sciences
2Department
of Pharmacology and Nephrology
University of Mississippi Medical Center, Jackson, MS 39216 Institution: University of Mississippi Medical Center, Jackson, MS Corresponding Author Bernadette E. Grayson University of Mississippi Medical Center Department of Neurobiology and Anatomical Sciences 2500 North State Street Jackson, MS 39216 Email: [email protected] Phone: 601-984-6809 Fax: 601-984-1655 Keywords: spinal cord injury, obese, high-fat diet, microarray ACKNOWLEDGEMENTS B.E.G is supported by the Department of Defense Award Number SC150016 and partially by the National Institute of General Medical Sciences Award Number P20GM104357. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health The work performed by M.R.G. through the UMMC Molecular and Genomics Facility is supported, in part, by funds from the National Institute of General Medical Sciences of the National Institutes of Health, including Mississippi INBRE (P20GM103476), Center for Psychiatric Neuroscience (CPN)-COBRE (P30GM103328) and Obesity, Cardio-renal and Metabolic Diseases-COBRE (P20GM104357).
1
Copyright © 2017 by the American Physiological Society.
ABBREVIATIONS ABCA1 ADH1 APOC4 ASGR1 CD68 DCKL2 GLP1R GPNMB HFD HMCGR HMOX1 HT2b IGF1 IGF2 LCAT MMP8 MMP12 PTGS1 tSCI TIMP1
ATP binding cassette subfamily A member 1 alcohol dehydrogenase 1 apolipoprotein C-IV asialoglycoprotein receptor 1 cluster of differentiation 68 doublecortin-like kinase 2, glucagon like peptide 1 receptor glycoprotein nmb high fat diet 3-hydroxy-3-methylglutaryl-CoA reductase heme oxygenase 1 serotonin receptor 2B insulin-like growth factor 1 insulin-like growth factor 2 lecithin cholesterol acyltransferase matrix metalloproteinase 8 matrix metalloproteinase 12 prostaglandin-endoperoxide synthase 1 thoracic spinal cord injury metallopeptidase inhibitor 1
2
ABSTRACT Individuals that suffer injury to the spinal cord can result in long-term, debilitating sequelae.
Spinal cord injured patients have increased risk for the development of
metabolic disease which can further hinder the effectiveness of treatments to rehabilitate the cord and improve quality of life. In the present study, we sought to understand the impact of high-fat diet induced obesity on spinal cord injury (SCI) by examining transcriptome changes in the area of the injury and rostral and caudal to site of damage 12 weeks after injury. Adult, male Long Evans rats received either thoracic level contusion of the spinal cord or sham laminectomy and then were allowed to recover on normal rat chow for 4 weeks and further on HFD for an additional 8 weeks. Spinal cord tissues harvested from the rats were processed for Affymetrix microarray and further transcriptomic analysis. Diverse changes in gene expression were identified in the injured cord in genes such as MMP12, APOC4, GPNMB and IGF1 and 2. The greatest signaling changes occurred in pathways involved in cholesterol biosynthesis and immune cell trafficking. Taken together, the cord changes in the chronically obese rat following thoracic spinal cord injury (SCI) injury reveal further potential targets for therapy. These could be further explored as they overlap with genes involved in metabolic disease.
3
INTRODUCTION To date, 275,000 Americans live with spinal cord injuries, with ~12,000 new cases per year (7). Approximately 45% of the injuries result in either complete or incomplete paraplegia from lesions of the thoracic, lumbar or sacral regions. Most impactful is that no effective treatments exist to permanently repair the injured spinal cord. Given the serious consequences of an injured spinal cord on every aspect of life, recovery of function and improvement of quality of life are imperative. Metabolic Syndrome (MetS) is a significant long-term problem for chronic spinal cord injury (SCI) patients (28, 30). MetS is a compilation of diseases which includes obesity, type-2 diabetes, dyslipidemia and cardiovascular disease (4). MetS may be exacerbated in the SCI population for a variety of reasons, including reduced or complete loss of physical activity (33). SCI individuals may be accelerated towards MetS due to change in body composition and physical activity coupled with altered calorie consumption (25, 35). However, when physical activity is statistically accounted for, specific components of MetS remain significant comorbidities of SCI (28, 30). SCI produces long-term motor, sensory and autonomic peripheral nervous system (PNS) dysregulation that may potentiate and exacerbate MetS in this population. Presently, over 2/3rds of the American population is overweight and about 1/3rd of the population is obese (10). Obesity is a significant component of MetS and in general is driven by modifiable life style factors which include consumption of calorie-dense, diets laden with saturated fats and refined carbohydrates (western type diet), reduced physical activity and exposure to chronic stress, among other risk factors. Obesity is a chronic lowgrade inflammatory disease characterized by an expansion of adipose tissue resulting in overall increased body weight (27). Due to disruption of the immune system as well as 4
for neurological and physiological reasons, obesity is associated with increased risk for diseases ranging from coronary artery disease to influenza, and complications during surgical procedures (13, 18, 24). Independently both SCI and MetS have serious long term ramifications. But taken together, a spinal cord injury coupled with MetS may negatively impact the trajectory of the SCI disease processes because of overlapping and integrated signaling systems common to both. Transcriptional changes after spinal cord injury have been extensively studied in lean animals (21, 31, 37), therefore, in the present study, we used a clinically-relevant rat model of spinal contusion injury coupled with diet-induced obesity to determine gene expression changes to the cord. Specifically, after either a thoracic spinal cord injury (tSCI) or sham laminectomy (sham), animals were allowed to recover on regular chow diet for 4 weeks and then provided a high-fat diet for an additional 8 weeks sufficient to produce obesity. Upon euthanasia, spinal cords were excised and microdissected. Cervical, thoracic and lumber level samples were processed for RNA. Affymetrix Microarray coupled with Genesifter© and Ingenuity Pathway Analysis© exploration of the data was performed on the thoracic compartment containing the lesion site with validation using samples from the three segments. A high degree of gene expression changes in cholesterol, inflammation and matrix protein pathways was observed. Additionally, it was also observed that trauma to the thoracic region of the spinal cord caused an altered gene profile in the uninjured cervical and lumbar regions. Taken together, thoracic level injury coupled with high-fat diet consumption causes significant durable transcriptome changes. METHODS
5
Animals: All procedures for animal use complied with the Guidelines for the Care and Use of Laboratory Animals by the National Institutes of Health and were reviewed and approved by the University of Mississippi Medical Center Institutional Animal Care and Use Committee. Male, Long Evans rats (400g) (Harlan, Indianapolis, IN) (N=10) were initially multiply housed and maintained in a room on a 12/12-h light/dark cycle at 25 °C and 5060% humidity with ad libitum access to water. Animals were maintained on standard chow (#8640, Envigo, 3.0 kCal/g; 17% fat, 54% carbohydrate, 29% protein).
Rats were
assigned to either sham-laminectomy (Sham) (N=5) or thoracic spinal cord injury (tSCI) (N=5) group in a counterbalanced fashion based on body weight on the day prior to the start of surgery. Surgery was performed and animals were allowed to recover for 4 weeks following surgery. Rats were singly housed from the time of surgery to the end of the study. Rats were switched to a palatable, high-fat diet (HFD) (#D03082706, Research Diets, New Brunswick, NJ, 4.54 kCal/g; 40% fat, 46% carbohydrate, 15% protein) for the remainder of the study totaling 8 weeks. Surgical procedures: All surgical procedures were performed on animals that were deeply anesthetized with an anesthetic cocktail (ketamine [80 mg/kg], xylazine [10 mg/kg], acepromazine [0.75 mg/kg]). tSCI surgeries were performed as previously described (29). Incisions were made on the animals’ dorsal skin and overlying muscles and the vertebral column was exposed. A laminectomy was performed at thoracic level 10 (T10) and the vertebral column was stabilized using forceps that grasp the ventral surface of the lateral spinous processes at vertebral levels T9 and T11. Using an Infinite Horizon Spinal Impactor Device (Precision Systems and Instrumentation, LLC, Fairfax
6
Station, VA), moderate contusion/compression injuries were delivered to the T10 spinal cord using 150 kdynes of force with a 1 sec dwell. The dura mater remained closed for the entire duration of SCI surgeries. Immediately following tSCI, the overlying muscles were sutured and the skin was securely closed using stainless steel wound clips. Sham surgeries were performed consisting of incisions to the animals’ dorsal skin exposing the musculature and vertebral column. A laminectomy was performed at thoracic level 10 (T10) and then the overlying muscles were sutured and the skin was securely closed using stainless steel wound clips. Postoperative animal care: Sham and tSCI rats received the following post-operative care with administration of: 1) an antibiotic (2.5mg/kg Baytril) once daily for a period of 10 days, 2) buprenorphine for post-surgical pain management (0.025 mg/kg, twice daily for a period of 5 days, then as needed); and 3) 3-5 mL of 0.9% saline, twice daily for a period of 3 days to ensure hydration. Beginning the day of tSCI surgery, each rat’s urinary bladder was manually expressed two to three times daily until the animal recovered the ability to void its bladder. Thoracic contusion results in disruption of the supraspinal pathways that are responsible for bladder voiding. In our hands, control of neurogenic bladder function returns in about 14 days. As a rule, bladder care was discontinued for an individual animal when it exhibited an already-voided bladder on two consecutive bladder care sessions. Body weight, composition: Following surgery, animals were weighed daily for the first 7 days and then weekly thereafter. Body lean and fat mass composition was analyzed using Echo Magnetic Resonance Imaging (echoMRI) (EchoMedical Systems, Houston,
7
TX) at 0 (time of surgery), 4 (start of HFD) and 11 wks (one week preceding the end of the experiment/euthanasia) . Tissue harvest: Twelve weeks following surgery, rats were euthanized by conscious decapitation starting at 6 h following the onset of the light cycle. The spinal cord was carefully excised using a rongeur instrument. The spinal cord was sectioned into three pieces measuring approximately 1.5 cm around each of the cervical, thoracic and lumbar enlargements. The T10 lesion site was completely included in the thoracic compartment. Tissue was flash frozen with methylbutane on dry ice and then stored in -80 ºC until further processing. Microarray analysis: Whole genome transcript analysis was performed using Affymetrix platforms from thoracic region spinal segments Sham and tSCI rats (n=4 per group). RNA was extracted using a QIAGEN miniprep RNA kit (QIAGEN, Inc, Valencia, CA) and evaluated for quality and integrity (Bio-Rad Experion™ System). All RNA gave RQI between 9-10. Spinal cord RNA were processed using manufacturer directions forGeneChip® 2.0 ST array and scanned using Affymetrix equipment (Scanner 3000 7G System). Hybridized chips were automatically washed, stained and scanned at the UMMC Institutional Molecular and Genomics Core using Affymetrix equipment. Data obtained from these gene expression studies are deposited in the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) with the GEO accession number XXXXXXX. Analysis of microarray data was performed using software provided by Affymetrix (Affymetrix® Expression Console™ Software) and commercially available GeneSifter™ software platform (http://www.genesifter.net). In brief, differentially expressed genes
8
evaluated by t-test using two methods: (1) FWER (family-wise error rate) procedure, p
