Genome-wide analysis of pain-, nerve- and neurotrophin -related gene expression in the degenerating human annulus

  • Helen E Gruber1, 2Email author,

    Affiliated with

    • Gretchen L Hoelscher1,

      Affiliated with

      • Jane A Ingram1 and

        Affiliated with

        • Edward N HanleyJr1

          Affiliated with

          Molecular Pain20128:63

          DOI: 10.1186/1744-8069-8-63

          Received: 15 February 2012

          Accepted: 18 August 2012

          Published: 10 September 2012

          Abstract

          Background

          In spite of its high clinical relevance, the relationship between disc degeneration and low back pain is still not well understood. Recent studies have shown that genome-wide gene expression studies utilizing ontology searches provide an efficient and valuable methodology for identification of clinically relevant genes. Here we use this approach in analysis of pain-, nerve-, and neurotrophin-related gene expression patterns in specimens of human disc tissue. Control, non-herniated clinical, and herniated clinical specimens of human annulus tissue were studied following Institutional Review Board approval.

          Results

          Analyses were performed on more generated (Thompson grade IV and V) discs vs. less degenerated discs (grades I-III), on surgically operated discs vs. control discs, and on herniated vs. control discs. Analyses of more degenerated vs. less degenerated discs identified significant upregulation of well-recognized pain-related genes (bradykinin receptor B1, calcitonin gene-related peptide and catechol-0-methyltransferase). Nerve growth factor was significantly upregulated in surgical vs. control and in herniated vs. control discs. All three analyses also found significant changes in numerous proinflammatory cytokine- and chemokine-related genes. Nerve, neurotrophin and pain-ontology searches identified many matrix, signaling and functional genes which have known importance in the disc. Immunohistochemistry was utilized to confirm the presence of calcitonin gene-related peptide, catechol-0-methyltransferase and bradykinin receptor B1 at the protein level in the human annulus.

          Conclusions

          Findings point to the utility of microarray analyses in identification of pain-, neurotrophin and nerve-related genes in the disc, and point to the importance of future work exploring functional interactions between nerve and disc cells in vitro and in vivo. Nerve, pain and neurotrophin ontology searches identified numerous changes in proinflammatory cytokines and chemokines which also have significant relevance to disc biology. Since the degenerating human disc is primarily an avascular tissue site into which disc cells have contributed high levels of proinflammatory cytokines, these substances are not cleared from the tissue and remain there over time. We hypothesize that as nerves grow into the human annulus, they encounter a proinflammatory cytokine-rich milieu which may sensitize nociceptors and exacerbate pain production.

          Keywords

          Low back pain Neurotrophins Nerves Microarray analysis

          Background

          Low back pain brings the patient to the spine surgeon, but the relationship between disc degeneration and pain production in the disc is still poorly understood. Patients with chronic low back pain do not have the leg pain which results when fragments of herniated disc push on nerves; instead, these patients experience pain that is thought to arise from the disc itself. Although some back pain may be related to lumbosacral anatomy/function spinal anatomy [1], age, and other factors (see Fairbank et al. for a recent systematic review of low back pain classification [2]), discogenic low back pain is not well understood [38]. As with all pain, the pain-initiating event results from complex cellular, molecular and functional events at the nociceptors (naked nerve endings) [9, 10]. Discogenic pain is believed to result from disc changes (possibly from outer annulus pressure on nerve endings, disc cell dysfunction, products of matrix degradation [11], or unknown events) which influence the nervous system by stimulation of annulus nociceptors.

          Recent studies focused over the last decade on molecular events and gene expression patterns related to pain [12], and studies have revealed that genome-wide gene expression studies provide a powerful methodology for identification of clinically relevant genes [13]. The objective of the present study was to perform a genome-wide analysis of annulus tissue from patients with discogenic back pain, compared to disc tissue from control subjects and herniated disc patients, in an analysis of the expression of pain, nerve and neurotrophin-related genes. Ontology searches for these specific topics were utilized in order to avoid searching large gene array data bases gene by gene, and because this technique provides a controlled vocabulary of search terms for gene characteristics [14].

          As cell-based therapies for disc degeneration progress, information on pain-, nerve- and neurotrophin-gene expression in disc tissue becomes increasingly important. Findings presented here have potential applications in future treatment modalities, such as perispinal administration of TNF-α inhibitors (such as etanercept/enbrel [15]) or use of specific small molecular antagonists to neurotrophins. New information presented here on the relationship between proinflammatory mediators, nerves and neurotrophins has the potential to contribute to future important antagonist profiles with application to discogenic back pain.

          Results

          Demographic features of the patient population are summarized in Table 1 which presents spinal site, grade, subject age, and whether surgical specimens were derived from herniated or non-herniated tissue. In the present work, gene expression analyses included three Thompson grade II and five grade III control disc specimens (obtained from Cooperative Human Tissue Network). Surgical specimens were analyzed from three grade II discs (two specimens of which were from herniated discs), four grade III discs (from herniated discs), five grade IV specimens (two of which were from herniated discs), and three grade V specimens (two of which were from herniated discs).
          Table 1

          Demographic data on disc tissues*

          Subject number

          Site*

          Thompson grade

          Age (years)/gender

          Herniated?

          Other information

          1

          Lumbar

          II

          34/F

          No

          Control

          2

          L3-L4

          II

          30/M

          No

          Control

          3

          L4-L5

          II

          54/F

          No

          Surgical specimen

          4

          Lumbar

          II

          34/F

          No

          Control

          5

          L5-S1

          II

          21/M

          Yes

          Surgical specimen; immuno **

          6

          L4-L5

          II

          40/F

          Yes

          Surgical specimen

          7

          L4-L5

          III

          36/F

          Yes

          Surgical specimen

          8

          C6-C7

          III

          45/F

          Yes

          Surgical specimen

          9

          L3-L4

          III

          52/F

          No

          Control

          10

          L3-L4

          III

          52/F

          No

          Control; immuno

          11

          L3-L4

          III

          52/F

          No

          Control

          12

          L3-L4

          III

          52/F

          No

          Control

          13

          L2-L3

          III

          33/F

          No

          Control

          14

          L5-S1

          III

          37/M

          Yes

          Surgical specimen; immuno

          15

          L4-L5

          III

          43/M

          Yes

          Surgical specimen; immuno

          16

          L5-S1

          IV

          63 F

          No

          Surgical specimen

          17

          L2-L3

          IV

          65/F

          No

          Surgical specimen

          18

          C5-C6

          IV

          59/M

          Yes

          Surgical specimen

          19

          L5-S1

          IV

          45/M

          Yes

          Surgical specimen; immuno

          20

          L5-S1

          IV

          43/M

          No

          Surgical specimen

          21

          L5-S1

          V

          72/F

          Yes

          Surgical specimen; immuno

          22

          L4-5

          V

          41/M

          No

          Surgical specimen; immuno

          23

          C6-7

          V

          57/F

          Yes

          Surgical specimen

          * Note that multiples specimens may have been derived from some subjects. L, lumbar; C, cervical; M, male; F, female; Control discs are normal discs obtained from the Cooperative Human Tissue Network.

          ** Indicates that specimens were utilized for selected immunohistochemical studies.

          The ontology gene expression analysis for pain and nerve categories included a variety of biological processes, molecular functions, and cellular components. To assist the reader in understanding these, Table 2 outlines the categories included in our searches.
          Table 2

          Outline of ontology search strategies

          Pain Ontologies:

          A. Biological Process:

          1. Sensory perception

           a. Sensory perception of pain:

            I. Regulation of sensory perception of pain

            II. Detection of chemical stimulus involved

            III. Detection of temperature stimulus involved

            IV. Detection of mechanical stimulus involved

          2. Ion transport

          3. Inflammatory response

          4. Behavior

          5. Response to pain:

           a. Behavioral response to pain

          6. G-protein coupled receptor (protein) signaling pathway

          7. Transmission of nerve impulse

          8. Fatty acid catabolic process

          B. Molecular Function:

          1. 1. Carboxyl- or carbamoyl-transferase activity

          2. Opioid peptide activity

          3. Signal transducer activity

           a. G-protein coupled receptor activity

           b. Galanin receptor activity

           c. Bradykinin receptor activity

           d. Opioid receptor activity

           e. Adrenergic receptor activity

          4. Voltage-gated channel activity

          5. G-protein coupled receptor binding

          C. Cellular component

          1. Plasma lipoprotein particle

          2. Axon

          Nerve Ontologies:

          A. Biological Process:

          1. Neuron death

           a. Neuron apoptosis

            i. Positive regulation of neuron apoptosis

            ii. Negative regulation of neuron apoptosis

           a. Neurotrophin production

            iii. Neuroprotection

          2. Neuropeptide signaling pathway

          3. Transmission of nerve impulse

          4. Nervous system development:

           a. Nerve development

           b. Neurogenesis

            i. Regulation of neurogenesis

             a. Positive regulation of neurogenesis

             b. Negative regulation of neurogenesis

            ii. Neuron differentiation

             a. Neuron projection development

            i. Axonogenesis regulation:

             a. Positive regulation of axonogenesis

             b. Negative regulation of axonogenesis

            iii. Neuroblast differentiation

            iv. Neuroblast proliferation

            v. Neuron migration

          B. Molecular function:

          1. Neurotrophin binding:

           a. Nerve growth factor binding

          2. Neurotrophin receptor activity

          3. Neurotransmitter receptor activity

           a. Substance P receptor activity

          4. Calcitonin receptor binding

          5. Vascular endothelial growth factor (-activated) receptor activity

          6. Protein kinase activity

          7. Neuropeptide hormone activity

          8. Neuropeptide binding

          9. Ciliary neurotrophic factor receptor binding

          10. Neurotrophin receptor binding:

           a. Nerve growth factor receptor binding

          C. Cellular component:

          1. Neuron projection:

           a. Axon

           b. Dendrite

          2. Endoplasmic reticulum lumen

          3. Nuclear outer membrane

          In each of our summary tables on gene expression findings (Tables  3, 4 and 5), genes are listed which have relevance to nerves, pain and neurotrophins (listed in section A of Tables  3, 4 and 5), genes with relevance to proinflammatory cytokines and chemokines (listed in section B of Tables  3, 4 and 5) and genes with specificity not only to nerve, pain and neurotrophins but also to the disc itself (listed in section C of the tables).
          Table 3

          Significant differences in pain-, neurotrophin-, nerve-, and disc-related genes in more degenerated discs (Grades iv and v) compared to less degenerated discs (Grades i – iii)

          Gene name

          Gene identifier

          Fold change

          Direction

          p-value

          A. Pain-, Neurotrophin- and Nerve-related Genes

          Adrenergic, beta, receptor kinase 2

          AI478542

          1.16

          Up

          0.045

          Ataxin 10

          AF119662

          2.62

          Up

          0.008

          Bradykinin receptor B1

          NM_000710

          1.09

          Up

          0.023

          Calcitonin gene related peptide

          AI478743

          1.51

          Up

          0.035

          Catechol-O-methyltransferase

          NM_001670

          1.17

          Up

          0.020

          Cholinergic receptor, muscarinic 1

          AI500293

          1.60

          Down

          0.042

          Clusterin

          M25915

          12.76

          Up

          0.010

          EGF, latrophilin and seven transmembrane domain containing 1

          NM_022159

          1.74

          Up

          0.040

          Endoplasmic reticulum aminopeptidase 2

          BE889628

          1.28

          Up

          0.036

          Endoplasmic reticulum protein 44

          BC005374

          2.08

          Up

          0.043

          GABA binding protein 2

          BC002557

          4.54

          Down

          0.027

          GABA(A) receptor-associated protein

          NM_007278

          3.78

          Up

          0.049

          Gap junction protein, alpha 1, 43 kDa

          NM_000165

          4.91

          Up

          0.017

          Glutathione peroxidase 1

          NM_000581

          3.42

          Up

          0.036

          Glutamate receptor, metabotropic 1

          U31216

          1.06

          Up

          0.0082

          Guanine nucleotide binding protein (G protein), gamma 10

          NM_004125

          2.97

          Up

          0.049

          Guanine nucleotide binding protein (G protein) alpha inhibiting activity polypeptide 3

          J03198

          3.22

          Up

          0.021

          Guanine nucleotide binding protein (G protein) beta polypeptide 1

          NM_002074

          4.04

          Up

          0.015

          Heat shock protein 90 kDa alpha (cytosolic), blass B member 1

          AF275719

          2.96

          Up

          0.038

          Hydroxysteroid (17-beta) dehydrogenase 4

          NM_000414

          1.88

          Up

          0.049

          Kv channel interacting protein 2

          AF367019

          1.52

          Up

          0.027

          Neurogenin 2

          AF022859

          1.08

          up

          0.045

          Neuron navigator 1

          N57538

          2.42

          Up

          0.009

          Neuron navigator 2

          AA011020

          1.11

          Up

          0.043

          Neuropilin 2

          AA295257

          1.87

          Up

          0.037

          Neuroplastin

          NM_017455

          2.78

          Up

          0.048

          Opioid growth factor receptor

          AF172449

          1.31

          Up

          0.008

          Palmitoyl-protein thioesterase 1

          NM_000310

          3.04

          Up

          0.019

          PDZ and LIM domain 5

          AV715767

          5.1

          Up

          0.006

          Peroxisome proliferator-activated receptor delta

          NM_006238

          3.31

          Down

          0.031

          Phytanoyl-CoA 2-hydroxylase

          NM_006214

          2.17

          Up

          0.049

          Potassium channel tetramerisation domain containing 10

          AS073741

          2.69

          Up

          0.036

          Potassium channel tetramerisation domain containing 15

          W73820

          1.33

          Up

          0.013

          Potassium voltage-gated channel, delayed-rectifier, subfamily S, member 3

          NM_002252

          1.66

          Up

          0.031

          Presenilin 1

          NM_007318

          1.61

          Up

          0.024

          Regulator of G-protein signaling 5

          AF159570

          2.31

          Up

          0.047

          Reticulon 4 (Neurite growth inhibitor)

          AF20999

          4.61

          Up

          0.001

          Solute carrier family 38, member 1

          NM_030674

          2.13

          Up

          0.039

          Solute carrier family 7 (catonic amino acid transporter, y + system), member 1

          AA148507

          1.44

          Up

          0.017

          Somatostatin receptor 4

          NM_001052

          1.09

          Down

          0.033

          S100 calcium binding protein A4

          NM_002961

          7.44

          Up

          0.010

          Thioredoxin domain containing 12 (endoplasmic reticulum)

          AF131758

          2/42

          Up

          0.009

          Thy-1 cell surface antigen

          AL161958

          3.48

          Up

          0.006

          B. Proinflammatory cytokine and chemokine genes:

          Chemokine (C-X-C motif) receptor 7

          AI817041

          3.32

          Up

          0.043

          Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)

          NM_000609

          1.90

          Up

          0.024

          FGF receptor 2

          T83672

          1,12

          Up

          0.020

          IL-17A

          NM_002190

          1.05

          Up

          0.048

          Interferon induced transmembrane protein 1 (9–27)

          NM_003641

          2.72

          Up

          0.026

          PDGF C

          NM_016205

          3.07

          Up

          0.041

          PDGF alpha polypeptide

          NM_006206

          5.73

          Up

          0.021

          PDGF receptor, beta polypeptide

          NM_003609

          2.10

          Up

          0.033

          PDGF receptor-like

          NM_006207

          1.82

          Up

          0.037

          TGF beta

          NM_000358

          6.68

          Up

          0.035

          Latent TGF beta binding protein 1

          NM_000627

          3.29

          Up

          0.016

          TNF receptor-associated factor 5

          NM_004619

          1.34

          Up

          0.014

          VEGF B

          NM_003377

          1.09

          Up

          0.037

          C. Genes with special disc relevance:

          ADAM metallopeptidase domain 17

          NM_003183

          1.56

          Up

          0.001

          Apoptosis-inducing factor, mitochondrion associated, 1

          NM_004208

          1.42

          Up

          0.019

          BMP receptor, type II (serine/threonine kinase)

          AI457436

          1.51

          Up

          0.027

          Calcitonin receptor-like

          AI1478743

          1.51

          Up

          0.035

          Caspase 2, apoptosis-related cysteine peptidase

          AU153405

          1.63

          Up

          0.028

          Caspase 6, apoptosis-related cysteine peptidase

          BC000305

          1.63

          Up

          0.048

          Collagen, type I, alpha 1

          K01228

          2.69

          Up

          0.046

          Collagen, type I, alpha 2

          NM_000089

          13.27

          Up

          0.007

          Collagen type III, alpha 1

          AU144167

          10.3

          Up

          0.036

          Collagen type IV, alpha 2

          X05610

          2.02

          Up

          0.021

          Collagen type V, alpha 1

          AI9833428

          1.5

          Up

          0.039

          Collagen type VI, alpha 3

          NM_004369

          5.85

          Up

          0.036

          Connective tissue growth factor

          M92934

          6.47

          Up

          0.009

          EGF-containing fibulin-like extracellular matrix protein 1

          NM_004105

          1.49

          Up

          0.005

          Fibronectin 1

          AK026737

          16.82

          Up

          0.017

          Fibronectin leucine rich transmembrane protein 2

          NM_013231

          2.78

          Up

          0.023

          Hypoxia inducible factor 1, alpha subunit

          NM_001530

          3.61

          Up

          0.037

          Janus kinase 2

          AF001362

          1.3

          Up

          0.002

          Laminen, alpha 5

          BC003355

          1.39

          Up

          0.042

          Laminin, beta 1

          NM_002291

          2.13

          Up

          0.045

          Lumican

          NM_002345

          17.58

          Up

          0.015

          Mitogen-activated protein kinase 1

          AF320999

          4.61

          Up

          0.0017

          Mitogen-activated protein kinase 13

          BC000433

          1.37

          Up

          0.014

          Mitogen-activated protein kinase kinase kinase 3

          BF971923

          1.67

          Up

          0.045

          Nitric oxide synthase 3

          NM_000603

          4.77

          Down

          0.030

          SPARC (osteonectin), cwcv and Kazal-like domains proteoglycan (testican) 1

          AF231124

          2.74

          Up

          0.027

          Thrombospondin 1

          BF055462

          1.20

          Up

          0.033

          TIMP metallopeptidase inhibitor 2

          BF107565

          3.57

          Up

          0.034

          TIMP metallopeptidase inhibitor 3

          U67195

          2.88

          Up

          0.035

          Vimentin

          AI520969

          1.35

          Up

          0.031

          Table 4

          Significant differences in pain-, neurotrophin-, nerve- and disc related gene expression in surgical compared to control (CHTN) specimens

          Gene name

          Gene identifier

          Fold change

          Direction

          p-value

          A. Pain-, Neurotrophin-, and Nerve-related genes:

          Adrenergic, beta, receptor kinase 1

          NM_001619

          1.28

          Down

          0.015

          Ataxin 1

          NM_000332

          1.89

          Up

          0.026

          Ataxin 10

          AF119662

          2.43

          Up

          0.016

          Caireticulin

          NM_004343

          8.53

          Up

          0.005

          Calcitonin gene-related peptide

          M26095

          1.21

          Down

          0.005

          Calcium channel, voltage-dependent, gamma subunit 2 (stargazin)

          NM_006078

          1.18

          Down

          0.037

          Calcium channel, voltage-dependent, P/Q type, alpha 1A subunit

          AA769818

          1.92

          Up

          0.023

          Calcium channel, voltage-dependent, beta 4 subunit

          NM_000726

          1.24

          Down

          0.001

          Calmodulin 3 (phosphorylase kinase, delta)

          NM_005184

          4.29

          Up

          0.002

          Calreticulin

          NM_004343

          8.53

          Up

          0.005

          Cannabinoid receptor 1 (brain)

          NM_001840

          1.14

          Down

          0.021

          Catechol-O-methyltransferase

          AW139431

          1.29

          Down

          0.035

          Chloride intracellular channel 1

          L08666

          3.61

          Up

          0.035

          Cholinergic receptor, muscarinic 1

          AI500293

          1.99

          Down

          0.001

          Ciliary neurotrophic factor

          NM_000614

          1.14

          Down

          0.040

          Clusterin

          AI982754

          20.62

          Up

          0.008

          Early growth response 1

          NM_001964

          5.73

          Up

          0.013

          Family with sequence similarity 123, member B

          NM_019000

          3.45

          Up

          0.022

          Frizzled homolog 8

          AB043703

          2.81

          Up

          0.016

          G protein-coupled receptor, family C, group 5, member C

          NM_022036

          2.32

          Up

          0.039

          G protein-coupled receptor 87

          NM_023915

          1.17

          Down

          0.006

          G protein-coupled receptor 98

          AF037334

          1.17

          Down

          0.043

          G protein regulated inducer of neurite outgrowth 1

          AI052709

          1.05

          Down

          0.046

          GABA binding protein 2

          BC002557

          8.78

          Down

          0.0006

          GABA(A) receptor-associated protein

          NM_007278

          4.04

          Up

          0.038

          GABA A receptor, beta 2

          NM_021911

          1.2

          Down

          0.006

          Gap junction protein, alpha 1, 43 kDa

          NM_000165

          4.1

          Up

          0.038

          Glutamate receptor, ionotropic, delta 2

          NM_001510

          1.17

          Down

          0.046

          Glutamate receptor, ionotropic, kainite 5

          S40369

          1.28

          Down

          0.009

          Glutamate receptor, ionotropic, N-methyl D-aspartate 2B

          NM_024016

          1.08

          Down

          0.017

          Glutamate receptor, metabotropic 5

          D60132

          1.38

          Down

          0.007

          Guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 3

          J03198

          2.87

          Up

          0.040

          Guanine nucleotide binding protein (G protein), beta polypeptide 1

          NM_002074

          4.36

          Up

          0.009

          Guanine nucleotide binding protein (G protein), alpha 11 (Gq class)

          NM_002067

          1.29

          Up

          0.024

          Glutathione peroxidase 1

          NM_000581

          3.62

          Up

          0.028

          Low density lipoprotein-related protein 1

          NM_002332

          5.26

          Up

          0.034

          Meteorin, glial cell differentiation regulator

          BE965311

          1.28

          Down

          0.049

          Natriuretic peptide receptor B/guanylate cyclase B

          NM_003995

          1.55

          Up

          J0.033

          Nerve growth factor, beta polypeptide

          NM_002506

          1.44

          Up

          0.03

          Netrin 1

          BF591483

          1.16

          Down

          0.041

          Neugrin, neurite outgrowth associated

          AL037339

          2.66

          Up

          0.028

          Neuroligin 1

          AI912122

          1.13

          Down

          0.043

          Neuron navigator 1

          AB032977

          3.83

          Up

          0.005

          Neuro-oncological ventral antigen 1

          NM_002515

          3.1

          Up

          0.0008

          Neuropilin 2

          BC009222

          3.28

          Up

          0.014

          Neuroplastin

          NM_017455

          3.61

          Up

          0.015

          Neurotensin

          NM_006183

          1.22

          Down

          0.009

          Nexin (serpin peptidase inhibitor, claude E)

          AL541302

          5.64

          Up

          0.009

          Oligophrenin 1

          NM_002547

          2.82

          Up

          0.048

          Opioid growth factor receptor-like 1

          BE500942

          2.4

          Up

          0.023

          Palmitoyl-protein thioesterase 1

          NM_000310

          3.64

          Up

          0.005

          PDZ and LIM domain 5

          AV15767

          5.06

          Up

          0.006

          Potassium large conductance calcium-activated channel, subfamily M, alpha member 1

          U11058

          3.44

          Up

          0.040

          Potassium voltage-gated channel, shaker-related subfamily, member 10

          NM_005549

          2.02

          Down

          0.010

          Potassium channel tetramerisation domain containing 20

          AV707142

          2.34

          Up

          0.018

          Prepronociceptin

          NM_006228

          1.04

          Down

          0.027

          Regulator of G-protein signaling 3

          NM_021106

          2.45

          Up

          0.039

          Regulator of G-protein signaling 12

          AF030111

          1.18

          Down

          0.006

          Regulator of G-protein signaling 18

          AF076642

          1.3

          Down

          0.025

          Reticulon 4 (Neurite growth inhibitor)

          AB015639

          5.96

          Up

          0.004

          Roundabout, axon guidance receptor, homolog 3

          NM_022370

          1.89

          Up

          0.025

          S100 calcium binding protein A4

          NM_002961

          12.85

          Up

          0.003

          S100 calcium binding protein A6

          NM_014624

          6.97

          Up

          0.013

          S100 calcium binding protein B

          BC001766

          3.48

          Up

          0.009

          Sigma non-opioid intracellular receptor 1

          NM_005866

          1.64

          Up

          0.031

          Solute carrier family 1 (glutamate/neutral amino acid transporter), member 4

          W72527

          4.06

          Up

          0.001

          Solute carrier family 29 (nucleoside transporters), member 1

          NM_004955

          2.89

          0.009

           

          Solute carrier family 38, member 1

          NM_030674

          2.39

          Up

          0.015

          Synaptopodin

          NM_007286

          2.24

          Up

          0.032

          Thioredoxin domain containing 5 (endoplasmic reticulum)

          NM_030810

          6.05

          Up

          0.002

          Thy-1 cell surface antigen

          AL161958

          3.13

          Up

          0.013

          Voltage-dependent anion channel 2

          L08666

          3.61

          Up

          0.035

          Xylosyltransferase 1

          AI693140

          6.23

          Up

          0.004

          B. Proinflammatory cytokine and chemokine genes:

          Chemokine (C-X-C motif) ligand 2

          AV648479

          1.15

          Down

          0.027

          Chemokine (C-X-C motif) receptor 7

          AI817041

          4.13

          Up

          0.014

          FGF 1 (acidic)

          X59065

          2.17

          Up

          0.033

          FGF 5

          AB016517

          1.47

          Down

          0.015

          FGF receptor substrate 2

          AI708648

          1.23

          Down

          0.027

          FGF receptor 3

          NM_000142

          2.62

          Up

          0.048

          FGF receptor substrate 2

          AI708648

          1.23

          Down

          0.027

          IL-1 receptor-associated kinase 1

          NM_001569

          3.98

          Up

          0.010

          IL-1 receptor-like 1

          NM_003856

          1.16

          Down

          0.041

          IL-31 receptor B1

          NM_139017

          1.17

          Down

          0.025

          IL-6 signal transducer (gp130, oncostatin M receptor)

          AW242916

          1.56

          Down

          0.032

          Interferon gamma receptor 2

          NM_005534

          2.25

          Up

          0.041

          Macrophate migration inhibitory factor

          NM_002415

          5.71

          Up

          0.004

          PDGF C

          NM_016205

          4.62

          Up

          0.003

          PDGF receptor, alpha polypeptide

          NM_006206

          6.63

          Up

          0.011

          TGF beta

          NM_000358

          8.77

          Up

          0.014

          TGF beta receptor 1

          AA604375

          3.14

          Up

          0.023

          TNF (ligand) superfamily, member 11

          AF053712

          3.28

          Up

          0.009

          TNF alpha-indued protein 6

          AW188198

          9.91

          Up

          0.006

          TNF receptor superfamily member 1A

          NM_001065

          2.01

          Up

          0.040

          TNF receptor superfamily, member 10c, decoy without an intracellular domain

          AK026079

          1.34

          Down

          0.014

          TNF receptor superfamily, member 10d, decoy without an intracellular domain

          NM_003841

          1.18

          Down

          0.039

          TNF receptor superfamily, member 12A

          NM_016639

          2.01

          Up

          0.046

          TNF receptor superfamily, member 25

          U94506

          1.16

          Down

          0.012

          TNF receptor-associatee factor 3

          AI721219

          1.57

          Up

          0.046

          C. Genes with special disc relevance:

          ADAM metallopeptidase domain 17

          NM_003183

          1.38

          Up

          0.025

          ADAM metallopeptidase domain 22

          NM_021723

          1.18

          Down

          0.045

          BCL2-associated athanogene 5

          NM_004873

          1.9

          Up

          0.028

          BMP and activin membrane-bound inhibitor homolog

          NM_012342

          3.28

          Up

          0.008

          Brevican

          AA622130

          1.30

          Down

          0.044

          Caspase 6, apoptosis-related cysteine peptidase

          BC000305

          2.05

          Up

          <0.0001

          Collagen type II, alpha 1

          X)6268

          53.83

          Up

          0.009

          EGF-containing fibulin-like extracellular matrix protein 2

          NM_005507

          11.88

          Up

          0.012

          FGF 5

          AB016517

          1.47

          Down

          0.015

          Fibronectin 1

          AK026737

          37.09

          Up

          0.001

          Growth arrest specific 1

          NM_002048

          5.44

          Up

          0.006

          Growth arrest specific 7

          BE439987

          2.56

          Up

          0.040

          Heat shock 70kDa protein 5

          AW052044

          1.34

          Up

          0.047

          Heat shock 70kDa protein 8

          AA704004

          3.99

          Up

          0.023

          Heat shock protein 90 kDa beta, (group 94), member 1

          AK025862

          2.45

          Up

          0.031

          Hypoxia inducible factor 1, alpha subunit

          NM_001530

          4.41

          Up

          0.014

          Laminin, beta 2

          X79683

          3.24

          Up

          0.029

          Latent TGF beta binding protein 1

          NM_000627

          3.09

          Up

          0.024

          Lumican

          NM_002345

          66.35

          Up

          0.0001

          Lysyl oxidase-like 2

          NM_002318

          5.06

          Up

          0.010

          Mitogen-activated protein kinase 1

          BF434653

          1.38

          Down

          0.018

          Mitogen-activated protein kinase 3

          X60188

          1.94

          Up

          0.030

          Mitogen-activated protein kinase kinase kinase 3

          BF971923

          1.83

          Up

          0.016

          Mitogen-activated protein kinase 13

          BC000433

          1.35

          Up

          0.020

          Mitogen-activated protein kinase 14

          AA604375

          3.14

          Up

          0.023

          Proteoglycan 4

          NM_005807

          13.72

          Up

          0.001

          PTH 1 receptor

          NM_000316

          1.77

          Up

          0.047

          Retinoic acid receptor, beta

          NM_000965

          1.61

          Up

          0.036

          SOD

          NM_000454

          4.18

          Up

          0.023

          SPARC/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 1

          AF231124

          3.00

          Up

          0.015

          Tenascin R

          Y13359

          1.38

          Down

          0.014

          Thyroid stimulating hormone receptor

          BE045816

          1.26

          Down

          0.019

          TIMP metallopeptidase inhibitor 3

          NM_000362

          12.35

          Up

          0.001

          TIMP metallopeptidase inhibitor 4

          NM_003256

          2.43

          Up

          0.042

          Versican

          BF218922

          5.19

          Up

          0.021

          Table 5

          Significant differences in pain-, neurotrophin-, nerve-related and disc genes in herniated discs compared to control (CHTN) discs

          Gene name

          Gene identifier

          Fold change

          Direction

          p-value

          A. Pain-, Neurotrophin-, Nerve-related Genes:

           

          Adrenergic, beta, receptor kinase 1

          NM-001619

          1.33

          Down

          0.042

          Ataxin 1

          NM_000332

          2.09

          Up

          0.031

          Calcitonin gene related peptide

          M26095

          1.2

          Down

          0.040

          Calcium channel, voltage-dependent, P/Q type, alpha 1A subunit

          AA769818

          2.17

          Up

          0.018

          Calcium channel, voltage-dependent, beta 2 subunit

          U80764

          1.21

          Up

          0.037

          Cannabinoid receptor 1 (brain)

          NM-001840

          1.16

          Down

          0.029

          Chemokine (C-X-C motif) receptor 7

          AI817041

          4.51

          Up

          0.012

          Cholinergic receptor, muscarinic 1

          AI500293

          2.05

          Down

          0.003

          Clusterin

          M25915

          20.02

          Up

          0.003

          Corticotropin releasing hormone receptor 1

          X72304

          1.53

          Up

          0.035

          EPH receptor B3

          NM_004443

          1.52

          Up

          0.037

          Family with sequence similarity 134, member B

          NM_019000

          3.72

          Up

          0.021

          GABA(A) receptor-associated protein

          NM_007278

          4.39

          Up

          0.039

          GABA A receptor, beta 2

          NM_021911

          1.21

          Down

          0.016

          G protein-coupled receptor, family C, group 5, member C

          NM_022036

          2.64

          Up

          0.036

          G protein-coupled receptor 52

          NM_005684

          1.06

          Down

          0.044

          G protein-coupled receptor 161

          AI703188

          1.71

          Up

          0.044

          G protein signaling modulator 2 (AGS3-like)

          AW195581

          1.94

          Up

          0.040

          Glutamate receptor, metabotropic 5

          D60132

          1.37

          Down

          0.026

          Glutaminase

          NM_014905

          3.14

          Up

          0.014

          Glutathione peroxidase 1

          NM_000581

          3.94

          Up

          0.038

          Guanine nucleotide binding protein (G protein), beta polypeptide 1

          NM_002074

          4.61

          Up

          0.013

          Guanine nucleotide binding protein (G protein), gamma 5

          NM_005274

          2.62

          Up

          0.040

          Guanine nucleotide binding protein (G protein), gamma 7

          AL039870

          1.54

          Up

          0.006

          Guanine nucleotide binding protein (G protein), gamma 10

          AI765321

          1.49

          Up

          0.040

          Guanine nucleotide binding protein (G protein), gamma 11

          NM_004126

          3.13

          Up

          0.026

          Guanine nucleotide binding protein (G protein), gamma 12

          N32508

          1.86

          Up

          0.026

          Guanine nucleotide binding protein (G protein), alpha 13

          AI928136

          2.80

          Up

          0.022

          Kallidrein-related peptidase 8

          NM_144506

          1.21

          Down

          0.028

          Monoglyceride lipase

          BG168471

          1.79

          Up

          0.019

          Myelin basic protein

          N37023

          3.28

          Up

          0.003

          Natriuretic peptide receptor B/guanylate cyclase B (atrionatriuretic peptide receptor B)

          NM_003995

          2.14

          Up

          0.049

          Neogenin homolog 1

          BF058828

          1.28

          Down

          0.022

          Nerve growth factor (beta polypeptide)

          NM_002506

          1.5

          Up

          0.029

          Neural precursor cell expressed, developmentally downregulated 4-like

          AB007899

          1.23

          Up

          0.038

          Neuromedin U receptor 2

          AF272363

          1.09

          Down

          0.023

          Neuro-oncological ventral antigen 1

          NM_002515

          3.1

          Up

          0.001

          Neurofibromin 1

          M60915

          1.04

          Up

          0.031

          Neuromedin U receptor 2

          AF272363

          1.09

          Down

          0.023

          Neuropilin 2

          AA295257

          3.92

          Up

          0.032

          Neuroplastin

          NM_017455

          4.46

          Up

          0.008

          Opioid growth factor receptor-like 1

          BE500942

          2.52

          Up

          0.017

          Palmitoyl-protein thioesterase 1

          NM_000310

          4.34

          Up

          0.005

          Pancreatic polypeptide receptor 1

          U42387

          1.31

          Down

          0.048

          PDZ and LIM domain 5

          AV715767

          5.04

          Up

          0.007

          Plexin A1

          T16388

          1.51

          Down

          0.046

          Potassium voltage-gated channel, shaker-related subfamily, member 10

          NM_005549

          2.36

          Down

          0.007

          Prostaglandin E receptor 2 (subtype EP2), 53 kDa

          NM_000956

          1.45

          Up

          0.048

          Regulator of G-protein signaling 3

          NM_021106

          3.15

          Up

          0.021

          Regulator of G-protein signaling 12

          AF030111

          1.18

          Down

          0.027

          Regulator of G-protein signaling 14

          NM_006480

          1.13

          Up

          0.042

          Reticulon 4 (neurite growth inhibitor)

          AB015639

          5.58

          Up

          0.007

          Roundabout, axon guidance receptor, homolog 3

          NM_022370

          1.74

          Up

          0.048

          S100 calcium binding protein A6

          NM_014624

          9.13

          Up

          0.017

          S100 calcium binding protein A9

          NM_002965

          1.93

          Up

          0.046

          S100 calcium binding protein B

          BC001766

          4.18

          Up

          0.012

          Sigma non-opioid intracellular receptor

          NM_005866

          2.01

          Up

          0.022

          Solute carrier family 1 (glutamate/neutral amino acid transporter), member 4

          AI889380

          2.79

          Up

          0.013

          Solute carrier family 6 (neurotransmitter transporter, creatine), member 8

          AI820043

          1.65

          Down

          0.021

          Solute carrier family 22, member 17

          NM_020372

          2.62

          Up

          0.044

          Solute carrier family 29 (nucleoside transporters), member 1

          NM_004955

          4.03

          Up

          0.010

          Solute carrier family 38, member 1

          NM_030674

          2.25

          Up

          0.023

          Solute carrier family 38, member 2

          NM_018976

          2.24

          Up

          0.048

          Solute carrier family 44, member 2

          AI264216

          1.88

          Up

          0.020

          Syntaxin 1A (brain)

          NM_004603

          2.46

          Up

          0.017

          Syntaxin binding protein 1

          NM_003165

          1.96

          Up

          0.049

          Thy-1 cell surface antigen

          NM_000633

          2.24

          Up

          0.025

          Xylosyltransferase I

          AI693140

          6.41

          Up

          0.007

          B. Proinflammatory cytokine and chemokine genes:

              

          Chemokine (C-C motif) ligand 22

          NM_002990

          1.10

          Down

          0.039

          IL 11 receptor, alpha

          NM_147162

          1.03

          Down

          0.049

          IL 17 receptor C

          BF196320

          1.11

          Down

          0.033

          IL 27 receptor, alpha

          AI983115

          1.19

          Down

          0.042

          IL 31 receptor A

          AF106913

          1.33

          Down

          0.040

          IL-1 receptor-associated kinase 1

          NM_001569

          4.30

          Up

          0.015

          IL-23, alpha subunit P19

          AJ296370

          1.62

          Up

          0.023

          Interferon gamma receptor 2

          NM_005534

          2.79

          Up

          0.028

          Latent TGF beta binding protein 1

          NM_000627

          2.61

          Up

          0.039

          Macrophage migration inhibitory factor

          NM_002415

          5.01

          Up

          0.034

          PDGF receptor, alpha polypeptide

          NM_006206

          5.96

          Up

          0.018

          TGF beta

          NM_000358

          12.34

          Up

          0.011

          TGF beta receptor 1

          NM_000714

          3.88

          Up

          0.018

          TNF alpha-induced protein 6

          AW188198

          14.57

          Up

          0.002

          TNF receptor superfamily, member 11b

          NM_002546

          3.31

          Up

          0.034

          TNF receptor superfamily, member 12A

          NM_016639

          2.29

          Up

          0.042

          TNF receptor superfamily, member 25

          U94506

          1.13

          Down

          0.046

          TNF receptor-associated factor 3

          AI721219

          1.67

          Up

          0.043

          TNF superfamily, member 11

          AF053712

          4.32

          Up

          0.002

          C. Genes with special relevance to the disc:

           

          ADAM metallopeptidase domain 17

          NM_003183

          1.36

          Up

          0.045

          BCL2-associated athanogene 5

          NM_004873

          1.95

          Up

          0.039

          Caspase 6, apoptosis-related cysteine peptidase

          BC000305

          2.48

          Up

          0.025

          Collagen type I, alpha 2

          NM_000089

          11.05

          Up

          0.017

          Collagen type II, alpha 1

          X06268

          58.92

          Up

          0.0001

          Collagen type III, alpha 1

          AU144167

          12.15

          Up

          0.026

          Collagen type VI, alpha 2

          AL531750

          3.14

          Up

          0.018

          Collagen type VI, alpha 3

          NM_004369

          8.55

          Up

          0.013

          Collagen type VI, alpha 1

          AA292373

          3.12

          Up

          0.037

          Collagen type XI, alpha 1

          NM_001854

          6.45

          Up

          0.020

          Collagen type IX, alpha 3

          NM_001853

          10.38

          Up

          0.002

          EGF receptor pathway substrate 8

          NM_004447

          1.55

          Up

          0.048

          EGF-containing fibulin-like extracellular matrix protein 2

          NM_005507

          9.96

          Up

          0.037

          Fibronectin 1

          AK026737

          52.43

          Up

          0.0004

          Fibronectin leucine rich transmembrane protein 2

          NM_013231

          2.61

          Up

          0.028

          Growth arrest-specific 1

          NM_002048

          5.45

          Up

          0.006

          Growth arrest-specific 7

          NM_005890

          2.04

          Up

          0.045

          Heat shock 70 kDa protein 8

          AA704004

          5.36

          Up

          0.008

          Hypoxia indicuble factor 3, alpha subunit

          NM_001530

          4.32

          Up

          0.027

          Hypoxia inducible factor 1 (HIF1A)

          NM_001530

          4.32

          Up

          0.027

          Jun oncogene

          NM_002228

          2.21

          Up

          0.027

          Lumican

          NM_002345

          79.38

          Up

          <0.0001

          Lysly oxidase-like 2

          NM_002318

          5.85

          Up

          0.009

          Mitogen activated protein kinase 1

          BF434653

          1.44

          Down

          0.022

          Mitogen-activated protein kinase 3

          X60188

          2.28

          Up

          0.023

          Mitogen-activated protein kinase 8

          AU152505

          1.41

          Up

          0.048

          Mitogen-activated protein kinase 13

          BC000433

          1.32

          Up

          0.030

          Mitogen-activated protein kinase 14

          NM_001315

          4.30

          Up

          0.006

          Never in mitosis gene a (NIMA)-related kinase 8

          AI9365173

          1.81

          Up

          0.006

          Never in mitosis gene a (NIMA)-related kinase 7

          AL080111

          2.43

          Up

          0.039

          Proteoglycan 4

          NM_005807

          18.05

          Up

          0.001

          SOD 1, soluble

          NM_000454

          4.44

          Up

          0.038

          SPARC/osteonectin, cwcv and kazal-like domains proteoclycan (testican) 1 (SPOCK1)

          AF231124

          3.3

          Up

          0.010

          Thyrotropin-releasing hormone receptor

          D16845

          1.25

          Down

          0.004

          TIMP metallopeptidase inhibitor 2

          BF107565

          2.9

          Up

          0.046

          TIMP metallopeptidase inhibitor 3

          NM_000358

          15.36

          Up

          0.002

          Versican

          BF218922

          6.59

          Up

          0.013

          Vitamin D receptor

          AA454701

          1.19

          Down

          0.039

          Neurotrophin-, nerve-, and pain-related gene, and disc gene expression patterns in the annulus: expression patterns in more degenerate compared to less degenerate discs

          In Table 3, findings are presented for selected relevant genes which were significantly elevated in expression in more degenerated Thompson grade IV and V discs compared to findings in grades I, II and III discs.

          Of major interest in Table 3 are a number of genes with high relevance to pain, neurotrophins and nerves. These include significantly upregulated expression in the more degenerated discs of the following: bradykinin receptor B1, calcitonin gene-related peptide, catechol-O-methyltransferase, neuron navigator-1 and −2, neuropilin 2, and reticulon 4 (also known as neurite growth inhibitor). These genes showed up regulation fold changes ranging from 1.09 to 4.61.

          A large number of genes in the ion transport grouping showed significant changes in this analysis; 69 genes showed significant up regulation, and 59 significant down regulation (most data not shown).

          Genes with specific high relationships to disc cell biology included these significantly upregulated genes in the more degenerated discs: apoptosis-inducing factor (mitochondrion associated), BMP receptor, type II, collagens type I, III, IV, V and VI, connective tissue growth factor, fibronectin, hypoxia inducible factor 1 (HIF1), several of the mitogen-activated protein kinases, SPARC (osteonectin), TGF-ß, lumican and TIMP metallopeptidase inhibitors-2 and −3. These genes showed upregulation fold changes ranging from 1.42 to 17.58. Nitric oxide synthase 3 showed a 4.77 fold downregulation.

          Neurotrophin-, nerve-, and pain-related gene, and disc gene expression patterns in the annulus: analysis of expression patterns in surgical compared to control (CHTN) specimens

          In Table 4, findings are presented for relevant genes which were significantly upregulated in surgically operated disc specimens compared to expression findings in control (CHTN) discs.

          Of high interest in Table 4 are a number of genes with high relevance to pain, neurotrophins and nerves. These include the following: calcitonin gene-related peptide, catechol-O-methyltransferase, ciliary neurotrophic factor, nerve growth factor, neuron navigator 1, neuropilin 2, reticulon 4, and roundabout axon guidance receptor. These genes showed up regulation fold changes ranging from 1.2 to 5.96.

          A large number of genes in the ion transport grouping also showed significant changes in this analysis; 87 genes showed significant up regulation, and 73 significant down regulation (most of the gene data in the ion transport group are not shown here).

          Genes with specific high relationships to disc cell biology included these significantly upregulated genes in surgical vs. control discs: heat shock proteins, fibronectin 1, versican, lumican, several of the mitogen-activated protein kinases, TIMP metallopeptidase inhibitors-3 and −4, TFG-ß, latent TGF binding protein 1, several of the TNF receptors, and collagen type II alpha 1. These genes showed up regulation fold changes ranging from 1.1 to 66.35. Notable down regulated genes included brevican and FGF 5 (with fold changes of 1.3 and 1.4, respectively).

          Neurotrophin-, nerve-, and pain-related gene, and disc gene expression patterns in the annulus: expression patterns in herniated compared to Non-herniated discs

          Although not related to discogenic low back pain, we were also interested in evaluating our data in terms of expression patterns which were significantly different in herniated discs vs. non-herniated discs (Table 5).

          Of high interest in Table 5 are a number of genes with high relevance to pain, neurotrophins and nerves. These include the following: Calcitonin gene related peptide (down regulated 1.2 fold). Upregulated genes included neuropilin 2, nerve growth factor, reticulon 4, roundabout axon guidance receptor; these genes were upregulated 1.5 to 5.58 fold.

          A large number of genes in the ion transport grouping also showed significant changes in this analysis; 98 genes showed significant up regulation, and 39 significant down regulation (most data not shown).

          Genes with specific high relationships to disc cell biology included these significantly upregulated genes: a number of the collagens, fibronectin 1, hypoxia inducible factors-1 and −2 (alpha subunit), latent TGF-ß binding protein 1, TGF-ß receptor 1, several of the mitogen-activated protein kinases, proteoglycan 4, SOD, and the apoptosis-associated genes caspase 6 and TNFRSF1A-associated via death domain.

          Immunohistochemical studies

          Paraffin-embedded annulus tissue was available for several of the subjects studied here (subjects # 5, 10, 14, 15, 19, 21 and 22 (Table 1) which enabled us to perform immunolocalization studies for products of three of the genes of special interest here (calcitonin gene-related peptide, catechol-0-methyltransferase and bradykinin receptor B1). For each of these immunolocalizations, cells were present with localization in single cells, clusters of cells, and both rounded and spindle-shaped cells in the outermost region of the annulus. Representative images are shown in Figures 1A-C; Figure 1D presents a negative control with the absence of any localization. Note that in Figures 1A-C adjacent cells were occasionally present which showed no localization.
          http://static-content.springer.com/image/art%3A10.1186%2F1744-8069-8-63/MediaObjects/12990_2012_Article_520_Fig1_HTML.jpg
          Figure 1

          Immunohistochemical localizations of calcitonin gene-related peptide, catechol-0-methyltransferase and bradykinin receptor B1: Representative images showing localization of calcitonin gene-related peptide (Figure 1A), catechol-0-methyltransferase (Figure 1B) and bradykinin receptor B1 (Figure 1C) in annulus regions of the human disc. Figure   1D illustrates a negative control. Arrows mark nearby cells which did not show localization.

          Discussion

          In the present study we performed a genome-wide microarray analysis of human annulus tissue from patients with discogenic back pain compared to disc tissue from control subjects or compared to and herniated disc patients we had a special focus upon analysis of the expression of pain, nerve and neurotrophin-related genes. Ontology searches were an efficient search technique for identification of pain, and nerve-related genes [14]. Although there is a large body of clinical literature on low back pain, molecular studies are few, and those in the literature primarily focus upon population-based genetic studies of polymorphisms (SNPs) (see [13] for a review of pain and spinal disease). To the best of our knowledge, the present work is the first (non-SNP) genome-wide study of pain, neurotrophin and nerve-related genes in disc degeneration.

          Pain-related genes

          Several well-recognized pain-related genes were found to have significant elevations in our analyses. Bradykinin receptor B1, calcitonin gene-related peptide and catechol-0-methyltransferase were significantly elevated in more degenerated discs (grade IV and V) compared to less degenerated (grades I-III) discs (Table 1). Bradykinin receptor B1 is formed after tissue injury and mediates hyperalgesia in chronic inflammation, but has very low expression in healthy tissue expression [16]. Calcitonin gene-related peptide (CGRP) has been found to be elevated in sensory nerves innervating inflamed tissue [17], in dorsal root ganglia and spinal cord in sciatic nerve injuries in the rat model by Orita et al. [18]. In the latter work, application or antibodies to nerve growth factor or its receptors TrkA or p75NTR blocked CGRP expression. Catechol-0-methyltransferase (COMT) codes for a protein which is important in catabolic pathways of a number of pain-relevant neurotransmitters, including noradrenalin, adrenaline and dopamine [19]. In the present analyses comparing surgically operated discs to control discs (Table 4) and herniated to control discs, calcitonin gene-related peptide was the only one of these genes which was significant, and in these comparisons it was downregulated 1.2-fold. The COMT gene is very interesting since patients with a specific polymorphism identified by Zubieta et al. showed higher sensory and affective pain ratings [20].

          Our ability to perform immunolocalization studies on human annulus tissues to determine the presence of bradykinin receptor B1, calcitonin gene-related peptide and catechol-0-methyltransferase (Figure 1) added strength to the present study, and confirmed the presence of products of these gene at the protein level within human disc tissue.

          Neurotrophin-related genes

          Neurotrophins were also identified in the present analyses, including nerve growth factor in our studies of surgical vs. control discs, and herniated vs. control discs (significantly upregulated, Tables  4 and 5, respectively), and ciliary neurotrophic factor (down regulated, Table 4, surgical vs. control discs).

          Studies have recently shown production of several neurotrophins by disc cells. Gigante et al. reported the presence of nerve growth factor (NGF) mRNA and the high affinity tyrosine kinase A receptor (trkA) and the low affinity p75 receptor in the rounded cells in the disc annulus and nucleus pulposus [21]. Recently Abe et al. reported on the expression of nerve growth factor (NGF) by human disc cells in control disc tissue in vivo and in vitro in cells from control discs using immunocytochemistry [22]. Nerve growth factor was found to be high in the outer annulus and herniated disc tissue. That work also demonstrated that the proinflammatory cytokines IL-1 and TNF-alpha had stimulatory effects on NGF. These authors suggested that such actions may play a role in nerve sprouting into the disc and may be associated with discogenic pain. Recent in vitro work from our lab has also confirmed that exposure of disc cells to IL-1ß in three-dimensional culture (which more accurately mimics the in vivo condition) results in elevated production of nerve growth factor by human annulus cells [23].

          Ciliary neurotrophic factor whose expression is reported here, has not previously been known to be expressed in the human disc. Work from previous studies has shown that it can act as both a neuroprotective agent [24] and a trophic factor for motor neurons [25].

          Nerve-related genes

          Several nerve-related genes should be mentioned in our analyses, including neuron navigator-1 and −2, neuropilin 2, reticulon 4 (neurite growth inhibitor), roundabout axon guidance receptor, homolog 3, and neural precursor cell expressed (developmentally down regulated 4-like) (Tables  3, 4 and 5). These genes also have not previously been known to be expressed in the human disc.

          Neuron navigator 1 is a microtubule-associated protein involved in neuronal migration [26], and neuron navigator 2 is required for all-trans retinoic acid-mediated neurite outgrowth and axonal elongation [27]. Neuropilin 2 was significantly upregulated in all of our disc comparisons (Tables  3 4 and 5). Neuropilin 2 is another gene which we have found to be significantly upregulated in cultured annulus cells exposed to IL-1ß [23]. Neuropilin-1 and neuropilin-2 are membrane proteins implicated in aspects of neurodevelopment. They are semaphorin III receptors as shown by the work of Kolodkin et al. [28], and are expressed in overlapping populations of neurons in the embryonic nervous system. Semaphorin III is a protein which, when secreted in vitro, results in the collapse of neuronal growth cones and chemorepulsion of neurites. It is also needed for correct sensory afferent innervation [29]. Expression of this gene by annulus cells may provide evidence that annulus cells express this gene to block neurite ingrowth into the disc.

          Roundabout (ROBO1) is a gene which encodes a receptor which is a member of the neural cell adhesion molecule family. It functions as an axon guidance receptor [30]. We found upregulation of this gene in surgical vs. control specimens (Table 4) and in herniated vs. control specimens (Table 5).

          Reticulon 4 (neurite growth inhibitor) was identified with upregulation in each of our analyses, with 4.6 fold (Table 3, more degenerated discs vs. less degenerated discs), 5.96 fold (Table 4, surgical vs. control discs) and 5.58 fold (Table 5, herniated vs. control discs) changes. This gene, also called NOGO, is a neurite outgrowth inhibitor [31].

          Genes related to proinflammatory cytokines and chemokines

          In Tables  3 4 and 5, in the section headed “Genes with special disc relevance” we have listed many proinflammatory cytokines and chemokines identified in our analyses. These deserve special mention here because a large number of proinflammatory cytokines are well recognized as products of disc cells themselves in vivo (see [3236] for an introduction to this field). Many chemokines are also produced by disc cells (our unpublished data). It is very important to note in the present study that many proinflammatory cytokines and chemokines are now known to induce or exacerbate inflammatory and neuropathic pain and hyperalgesia [3739]. We suggest here that this is an exceptionally important aspect of low back pain that has here-to-fore been little recognized. The degenerating human disc is primarily an avascular tissue site into which disc cells have contributed high levels of proinflammatory cytokines which are not cleared from the tissue and remain there over time. We suggest that as nerves grow into the human annulus, they encounter a proinflammatory cytokine-rich milieu which exacerbates pain production.

          Kim et al. using in vitro work has suggested that disc cells themselves are involved in inflammatory activities, and suggested that interactions between annulus cells and nerve cells enhances the production of growth factors responsible for neovascularization and nerve ingrowth into the disc [40]. Previous research by Aoki et al. showed that disc inflammation potentially promoted axonal regeneration of dorsal root ganglion neurons innervating the disc in a rat model [41]. Using gene correlations, recent work by Lee et al. suggests that IL-1ß is generated during degeneration of the disc, and this stimulates expression of agents such as nerve growth factor, which result in nerve in growth into the disc [42].

          It is important to recognize that proinflammatory cytokine production within the degenerating disc can also be exacerbated by repeated disc injury, which may lead to persistent proinflammatory cytokine elevation [43]; thus repeated disc injury may also influence neuroinflammation and pain.

          A final important comment from the joint literature on proinflammatory mediators concerns the fact that release of these agents in damaged tissue and in the spinal cord is known to sensitize the peripheral terminals of nociceptors [44]. It is possible that similar hyper excitability of pain transmitting neurons results from proinflammatory cytokines in the disc matrix during degeneration; proinflammatory cytokines likely to be at play here are IL-1ß and TNF-α.

          Genes which share high importance to the disc itself

          It was interesting that many genes were identified in our analyses which have relevance to the nerve, neurotrophin and pain ontology and to disc biology itself (as shown in section C of Tables  3 4 and 5). These included extracellular matrix (ECM) components, such as collagens, fibronectin, laminin, thrombospondin, brevican, proteoglycans, and versican, and also genes related to matrix degradation (metallopepdiases and TIMP metallopeptidase inhibitors), growth factors (connective tissue growth factor), nitric oxide synthase 3 (ENOS), SOD, and hypoxia inducible factor 1 alpha subunit. Also important to disc biology were the vitamin D receptor gene, growth arrest specific genes (important to cell senescence), apoptosis-related genes, and BMP receptor type II. Although some of these gene products may be resident only in sites of neurovascular ingrowth, many may influence the disc ECM itself and disc cell functions. Such findings point to the importance of future research directed towards identifying functional interactions between disc and nerve cells in vivo and in vitro. For example, it has long been recognized that there is an accumulation of fibronectin fragments in the aging/degenerating disc, and these fragments initiate signaling pathways which can increase MMP expression causing a cycle of matrix destruction [4550]. Strong up regulation of fibronectin was present in our analyses (16.8 fold upregulation in more degenerated vs. healthier discs (Table 3), 37.0 fold upregulation in surgical vs. control discs (Table 4), and a 52.4 fold up regulation in herniated vs. control discs (Table 5).

          Possible limitations to the present analyses include the fact that some specimens, noted as “controls” in Table 1, were obtained from the Cooperative Human Tissue Network (CHTN). Although these were shipped quickly to our lab post-autopsy as quickly as possible via CHTN, delays might result in potential alterations in mRNA levels during our study. The reader should note that in order to carefully investigate this issue, in Table 4 we compared findings in surgical specimens vs. those obtained from CHTN. As would be expected in different sized microarray group comparisons with ontology searchers, Table 4 does differ in some respects from data presented in Table 3. It is also worth noting that in our laboratory, CHTN specimens are also routinely used for derivation of disc cells in vitro, and in our hands over 98% of CHTN specimens yielded viable cells.

          Another point for the reader to note is that our tables have reported gene expression fold changes which in some cases were less than 2.0, a level which is commonly used. Since we feel that smaller changes in important genes may be clinically relevant, we have reported these changes in our data tables.

          Conclusions

          Even though our three study groups were not large, the present analysis showed that microarray analysis could successfully be used to examine key pain-, neurotrophin- and nerve-related genes in specimens of human disc tissue. Many genes were found in these ontology searches which held significance not only for nerves, pain and neurotrophins, but also for disc ECM, signaling and functional components. Key findings included confirmation of the presence of calcitonin gene-related peptide, catechol-0-methyltransferase and bradykinin receptor B1 at the protein level in the human annulus using immunohistochemistry, and identification of significant changes in a number of proinflammatory and chemokine genes identified from nerve, neurotrophin and pain ontology searches. Since the disc is primarily avascular, and since disc cells themselves produce proinflammatory cytokines and chemokines which are not removed from the tissue, we hypothesize that as nerves grow into the human annulus, they encounter a proinflammatory cytokine-rich milieu which may sensitize nociceptors and exacerbate pain production. Findings reported here point to the importance of future studies of the functional interactions between disc and nerve cells in vivo and in vitro.

          Methods

          Clinical study population

          Experimental study of human disc specimens was approved prospectively by the authors’ Human Subjects Institutional Review Board. The need for informed consent was waived by the ethical board since disc tissue was removed as part of routine surgical practice. Scoring of disc degeneration utilized a modification of the Thompson scoring system [51] incorporating author ENH’s radiologic, MRI and surgical findings. The Thompson system scores disc degeneration over the spectrum from a healthy disc (Thompson grade I) to discs with advanced degeneration (grade V, the most advanced stage of degeneration) [51]. Patient specimens were derived from surgical disc procedures performed on individuals with herniated discs and degenerative disc disease. Surgical specimens were transported to the laboratory in sterile tissue culture medium. Non-surgical, control donor disc specimens were obtained via the National Cancer Institute Cooperative Human Tissue Network (CHTN); they were shipped overnight to the laboratory in sterile tissue culture medium and processed as described below. Specimen procurement from the CHTN was included in our approved protocol by our human subjects Institutional Review Board.

          Microarray analysis

          Disc tissue was snap frozen in liquid nitrogen, pulverized (BioPulverizer, BioSpec Products, Inc., Bartlesville, OK, USA), and homogenized via the FastPrep-24 instrument (MP Biomedicals L.L.C., Santa Ana, CA, USA). Total RNA was isolated via a modified version of TRIzol Reagent (Life Technologies: Invitrogen, Carlsbad, CA, USA), and prepared for microarray hybridization using the GeneChip 3’ IVT Express Kit (Affymetrix, Santa Clara, CA). In brief, total RNA was reverse transcribed to synthesize cDNA, converted to double stranded DNA, subjected to transcription generating biotin-labeled amplified RNA (cRNA) and hybridized to the DNA microarray in the Affymetrix Fluidics Station 400. Affymetrix human U133 X3P arrays were used. The GCOS Affymetrix GeneChip Operating System (version 1.2, Affymetrix, Santa Clara, CA 95051) was used for determining gene expression levels. mRNA from annulus tissue from each subject was analyzed separately (i.e., samples were not pooled).

          Statistical analysis

          The GCOS Affymetrix GeneChip Operating System (version 1.2, Affymetrix, Santa Clara, CA) was used for determining gene expression levels. GeneSifterTM web-based software (VizX Labs, Seattle, WA, USA) was used to analyze all microarray data. Using GC-RMA (Robust multi-array average), Affymetrix ‘.cel’ files were uploaded to the GeneSifterTM web site and normalized, and corrected for false discovery rate (FDR). Using the student t-test (2 tailed, unpaired), statistical significance was determined (p < 0.05).

          Gene Ontology (GO) searches were employed in our analyses to select genes of interest and groups of critically important genes. This approach lets one avoid searching through results gene by gene, and provides a controlled vocabulary of search terms for gene characteristics. In our analyses, Gene Ontologies (GO) were generated by GeneSifterTM based on the Gene Ontology Consortium. Searches were performed in the present study on “pain” and “nerve”; for each, ontologies were searched under “biological process”, “molecular function” (the activities of the gene product at the molecular level), and “cellular component” (parts or cells or the extracellular milieu). To aid the reader in visualizing the key terms covered in these ontology grouping, details are provided in Table 2.

          Gene array data for the human disc specimens analyzed here have been uploaded to the Gene Expression Omnibus (GEO) website [GEO:GSE23130] and may be accessed there.

          Immunohistochemistry

          Bradykinin receptor B1 (BDKRB1) and calcitonin gene related peptide (CGRP) Immunohistochemistry: Disc specimens were fixed in 10% neutral buffered formalin, embedded undecalcified, paraffin sections cut at 4 μm, collected on PLUS slides(Cardinal Health, Dublin, OH) and dried at 60°C. Sections were deparaffinized in xylene (Cardinal) and rehydrated through graded alcohols (AAPER, Shelbyville, KY) to distilled water. Antigen retrieval was performed using Biocare Antigen Decloaker Solution, pH 6.0 (Biocare Medical, Concord, CA) for 20 minutes at 95°C followed by cooling for 20 minutes. The remainder of the procedure was performed using the Dako Autostainer Plus (Dako, Carpenteria, CA) automated stainer. Endogenous peroxidase was blocked using 3% H202 (Sigma, St Louis, MO). Slides were incubated for 30 minutes with Serum-Free Protein Block (Dako); blocking solution was drained from slides and primary antibody applied. Sections were incubated for one hour with anti-Bradykinin receptor B1 (BDKRB1) (Novus Biologicals, Littleton, CO) at a 1:50 dilution, or with for one hour with anti-calcitonin gene related peptide (CGRP) (Abcam, Cambridge, MA) at a 1:100 dilution. Secondary antibody was 4 + Biotinylated Universal Goat Link (Biocare) for 10 minutes followed by 4+ streptavidin HRP Label (Biocare) for 10 minutes and DAB (Dako) for 5 minutes. Slides were removed from stainer, rinsed in water, counterstained with light green, dehydrated, cleared and mounted with resinous mounting media. Universal Rabbit Negative (Dako, Carpinteria, CA) was used as a negative control.

          Catechol-O-methyltransferase (COMT) immunohistochemistry did not require antigen retrieval. Sections were prepared as described above, and incubated for one hour with anti-catechol-O-methyltransferase (COMT) (Lifespan Biosciences, Seattle, WA) at a 1:200 dilution. The secondary antibody and negative control utilized were as described above.

          Positive control human tissues were also included with each immunolocalization run; for bradykinin receptor B1 this was brain, for calcitonin gene related peptide this was thyroid, and for catechol-O-methyltransferase, adrenal.

          Abbreviations

          GO: 

          Gene ontologies

          GC-RMA: 

          Robust multi-array average

          HIF1: 

          Hypoxia inducible factor 1

          TGF-ß: 

          Transforming growth factor beta

          TIMP: 

          Tissue inhibitor of metalloproteinases

          CHTN: 

          Cooperative Human Tissue Network

          TNFα: 

          Tumor necrosis factor-alpha

          SNP: 

          Single nucleotide polymorphism

          CGRP: 

          Calcitonin gene-related peptide

          COMT: 

          Catechol-0-methyltransferase

          NGF: 

          Nerve growth factor

          IL-1: 

          Interleukin-1

          SOD: 

          Superoxide dismutase

          ECM: 

          Extracellular matrix.

          Declarations

          Acknowledgements

          The authors wish to than the Brooks Center for Back Pain Research for general laboratory support. We thank Synthia Bethea for expert technical assistance in mRNA isolation and processing, Nury Steuerwald, Ph.D. (Director) and Judy Vachris in the Molecular Biology Core for excellent assistance with microarray processing, and Natalia Zinchenko for expert assistance with histology.

          Authors’ Affiliations

          (1)
          Department of Orthopaedic Surgery, Carolinas Medical Center
          (2)
          Orthopaedic Research Biology, Cannon Research

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          This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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