Open Access

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

  • Helen E Gruber1, 2Email author,
  • Gretchen L Hoelscher1,
  • Jane A Ingram1 and
  • Edward N HanleyJr1
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.
https://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

References

  1. Vora AJ, Koerr KD, Wolfer LR: Functional anatomy and pathophysiology of axial low back pain: Disc, posterior elements, sacroiliac joint, and associated pain generators. Phys Med Rehabil Clin N Am 2010, 21: 679–709. 10.1016/j.pmr.2010.07.005View ArticlePubMed
  2. Fairbank J, Gwilym SE, France JC, Daffner SD, Dettori J, Hermsmeyer J, et al.: The role of classification of chronic low back pain. Spine 2012, 36: S19-S42.View Article
  3. Garcia-Cosamalon J, del Valle ME, Calavia MG, Garcia-Suarez O, Lopez-Muniz A, Otero J, et al.: Intervertebral disc, sensory nerves and neurotrophins: who is who in discogenic pain? J Anat 2010, 217: 1–15. 10.1111/j.1469-7580.2010.01227.xPubMed CentralView ArticlePubMed
  4. Hurri H, Karppinen J: Discogenic pain. Pain 2004, 112: 225–228. 10.1016/j.pain.2004.08.016View ArticlePubMed
  5. Hyodo H, Sato T, Sasaki H, Tanaka Y: Discogenic pain in acute nonspecific low-back pain. Eur Spine J 2005, 14: 573–577. 10.1007/s00586-004-0844-8PubMed CentralView ArticlePubMed
  6. Aoki Y, Takahashi K, Ohtori S, Moriya H: Scientific basis of interventional therapies for discogenic pain: Neural mechanisms of discogenic pain. In Spinal Reconstruction - Clinical Examples of Applied Basic Science, Biomechanics, and Engineering. Edited by: Lewandowski KUYMJ, Kalfas IH, Park P, McLain RF, Trantolo DJ. Informa Healthcare, New York; 2007:219–236.View Article
  7. Zhou Y, Abdi S: Diagnosis and minimally invasive treatment of lumbar discogenic pain - A review of the literature. Clin J Pain 2006, 22: 468–481. 10.1097/01.ajp.0000208244.33498.05View ArticlePubMed
  8. Peng B, Wu W, Hou S, Li P, Zhang C, Yang Y: The pathogenesis of discogenic low back pain. J Bone Joint Surg Br 2005, 87B: 62–67.
  9. Tauben D: Central nervous system modulation of spinal pain: Pain sensitization and implications for surgical selection. SpineLine 2005, 7–12.
  10. McHugh JM, McHugh WB: Pain: Neuroanatomy, chemical mediators, and clinical implications. AACN Clin Issues Adv Pract Acute Crit Care 2000, 11: 168–178. 10.1097/00044067-200005000-00003View Article
  11. Wuertz K, Stachi E, Boos N: The influence of matrix degradation products on pain development in the intervertebral disc. Eur Spine J 2008, 17: 1555. Abstract
  12. Gu J, Zhuo M, Caterina M, MacDermott AB, Malmnbert A, Neugebauer V, et al.: Molecular pain, a new era of pain research and medicine. Mol Pain 2005, 1: 1. 10.1186/1744-8069-1-1PubMed CentralView ArticlePubMed
  13. Kim DH, Schwartz CE: The genetics of pain: implications for evaluation and treatment of spinal disease. Spine J 2010, 10: 827–840. 10.1016/j.spinee.2010.05.013View ArticlePubMed
  14. The Gene Ontology Consortium: Gene ontology: tool for the unification of biology. Nature Genet 2000, 25: 25–29. 10.1038/75556PubMed CentralView Article
  15. Tobinick EL, Britschgi-Davoodifar S: Perispinal TNF-alpha inhibition for discogenic pain. Swiss Med Weekly 2003, 133: 170–177.
  16. Dray A, Perkins M: Bradykinin and inflammatory pain. Trends Neurosci 1993, 16: 99–104. 10.1016/0166-2236(93)90133-7View ArticlePubMed
  17. Donnerer J, Schuligal R, Stein D: Increased content and transport of substance P and calcitonin gene-related peptide in sensory nerves innervating inflamed tissue: evidence for a regulatory function of nerve growth factor in vivo. Neurosci 1992, 49: 693–698. 10.1016/0306-4522(92)90237-VView Article
  18. Orita S, Ohtori S, Nagata M, Horii M, Yamashita M, Yamauchi K, et al.: Inhibiting nerve growth factor or its receptors downregulates calcitonin gene-related peptide expression in rat lumbar dorsal roog ganglia innervating injured intervertebral discs. J Orthop Res 2010, 28: 1614–1620. 10.1002/jor.21170View ArticlePubMed
  19. Anderson S, Skorpen F: Variation in the COMT gene: implications for pain perception and pain treatment. Pharmaco Economics 2009, 10: 669–684.View Article
  20. Zubieta J-K, Heitzeg MM, Smith YR, Bueller JA, Xu K, Xu Y, et al.: COMT val158met genotype affects u-opioid neurotransmitter responses to a pain stressor. Science 2003, 299: 1240–1243. 10.1126/science.1078546View ArticlePubMed
  21. Gigante A, Bevilacqua C, Pagnotta A, Manzotti S, Toesca A, Greco F: Expression of NGF, Trka and p75 in human cartilage. Eur J Histochem 2003, 47: 339–344.PubMed
  22. Abe Y, Akeda K, An HS, Aoki Y, Pichika R, Muehleman C, et al.: Proinflammatory cytokines stimulate the expression of nerve growth factor by human intervertebral disc cells. Spine 2007, 32: 635–642. 10.1097/01.brs.0000257556.90850.53View ArticlePubMed
  23. Gruber HE, Hoelscher GL, Bethea S, Hanley EN: Interleukin 1b exposure significantly upregulates neurotrophin gene expression for brain-derived neurotrophic factor, neurotrophin 3 and neuropilin 2 during 3D culture of human annulus cells. Spine J 2011,11(10S):33S-34S.View Article
  24. Emerich DF, Winn SR, Hantraye PM, Peschanski M, Chen C-Y, McDermott PBEE, et al.: Protective effect of encapsulated cells producing neurotrophic factor CNTF in a monkey model of Huntington's disease. Nature 1997, 386: 395–399. 10.1038/386395a0View ArticlePubMed
  25. Barbin GG, Manthorpe MM, Varon SS: Purification of the chick eye ciliary neuronotrophic factor. J Neurochem 1984, 43: 1468–1478. 10.1111/j.1471-4159.1984.tb05410.xView ArticlePubMed
  26. Martinez-Lopez MJ, Alcantara S, Mascarno C, Perez-Branguli F, Ruiz-Lozano P, Maes T, et al.: Mouse neuron navigator 1, a novel microtubule-associated protein involved in neuronal migration. Mol Cell Neurosci 2005, 28: 599–612. 10.1016/j.mcn.2004.09.016View ArticlePubMed
  27. Muley PD, McNeill EM, Marzinke MA, Knobel KM, Barr MM, Clagett-Dame M: The atRA-responsive gene neuron navigator 2 functions in neurite outgrowth and axonal elongation. Dev Neurobiol 2008, 68: 1441–1453. 10.1002/dneu.20670PubMed CentralView ArticlePubMed
  28. Kolodkin AL, Levengood DV, Rowe EG, Tai Y-T, Giger RJ, Ginty DD: Neuropilin is a semaphorin III receptor. Cell 1997, 90: 753–762. 10.1016/S0092-8674(00)80535-8View ArticlePubMed
  29. Tessier-Lavigne M, Goodman CS: The molecular biology of axon guidance. Science 1996, 274: 1123–1133. 10.1126/science.274.5290.1123View ArticlePubMed
  30. Kidd T, Brose K, Mitchell KJ, Fetter RD, Tessier-Lavigne M, Goodman CS, et al.: Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionarily conserved guidance receptors. Cell 1998, 92: 205–215. 10.1016/S0092-8674(00)80915-0View ArticlePubMed
  31. Spillmann AA, Bandtlow CE, Lottspeich F, Keller F, Schwab ME: Identification and characterization of a bovine neurite growth inhibitor (bNI-220). J Biol Chem 1998, 273: 19283–19293. 10.1074/jbc.273.30.19283View ArticlePubMed
  32. Akyol S, Senel Eraslan B, Etyemez H, Tanriverdi T, Hanci M: Catabolic cytokine expressions in patients with degenerative disc disease. Turkish Neurosurg 2010, 20: 492–499.
  33. Hoyland JA, Le Maitre C, Freemont AJ: Investigation of the role of IL-1 and TNF in matrix degradation in the intervertebral disc. Rheumatol 2008, 47: 809–814. 10.1093/rheumatology/ken056View Article
  34. Shamji MF, Setton LA, Jarvin W, So S, Chen J, Jing L, et al.: Proinflammatory cytokine expression profiles in degenerated and herniated human intervertebral disc tissue. Arthritis Rheum 2010, 62: 1974–1982.PubMed CentralPubMed
  35. Gabr MA, Jing L, Helbling AR, Sinclair M, Allen KD, Shamji MF, et al.: Interleukin-17 synergizes with IFNg or TNFa to promote inflammatory mediator release and intercellular adhesion molecule-1 (ICAM-1) expression in human intervertebral disc cells. J Orthop Res 2011, 29: 1–7. 10.1002/jor.21206PubMed CentralView ArticlePubMed
  36. LeMaitre CL, Hoyland JA, Freemont AJ: Catabolic cytokine expression in degenerate and herniated human intervertebral discs: IL-1ß and TNFa expression profile. 2007. http://​arthritis-research.​com/​content/​9/​4/​R77
  37. Sommer C, Kress M: Recent findings on how proinflammatory cytokines cause pain: peripheral mechanisms in inflammatory and neuropathic hyperalgesia. Neurosci Lett 2004, 361: 184–187. 10.1016/j.neulet.2003.12.007View ArticlePubMed
  38. Boddeke EW: Involvement of chemokines in pain. Eur J Pharmacol 2001, 429: 115–119. 10.1016/S0014-2999(01)01311-5View ArticlePubMed
  39. Dickerson C, Undem B, Bullock B, Winchurch RA: Neuropeptide regulation of proinflammatory cytokine responses. J Leukocyte Biol 1998, 63: 602–605.PubMed
  40. Moon HJ, Kim JH, Lee HS, Chotai S, Kang JD, Suh JK, Park YK: Annulus fibrosus cells interact with neuron-like cells to modulate production of growth factors and cytokines in symptomatic disc degeneration. Spine 2012, 37: 2–9. 10.1097/BRS.0b013e31820cd2d8View ArticlePubMed
  41. Aoki Y, Ohtori S, Ino H, Duoya H, Ozawa T, Saito T, et al.: Disc inflammation potentially promotes axonal regeneration of dorsal root ganglion neurons innervating lumbar intervertebral disc in rats. Spine 2004, 29: 2621–2626. 10.1097/01.brs.0000146051.11574.b4View ArticlePubMed
  42. Lee JM, Song JY, Baek M, Jung H-Y, Kang H, Han IB, et al.: Interleukin-1b induces angiogenesis and innervation in human intervertebral disc degeneration. J Orthop Res 2011, 29: 265–269. 10.1002/jor.21210View ArticlePubMed
  43. Ulrich JA, Liebenberg EC, Thuillier DU, Lotz JC: ISSLS Prize Winner: Repeated disc injury causes persistent inflammation. Spine 2007, 32: 2812–2819. 10.1097/BRS.0b013e31815b9850View ArticlePubMed
  44. Schaible HG, Schmelz M, Tegeder I: Pathophysiology and treatment of pain in joint disease. Adv Drug Deliv Rev 2006, 58: 323–342. 10.1016/j.addr.2006.01.011View ArticlePubMed
  45. Oegema TRJ, Johnson SL, Aguiar DJ, Ogilvie JW: Fibronectin and its fragments increase with degeneration in the human intervertebral disc. Spine 2000, 25: 2742–2747. 10.1097/00007632-200011010-00005View ArticlePubMed
  46. Nerlich AG, Bachmeier BE, Boos N: Expression of fibronectin and TGF-b1 mRNA and protein suggest altered regulation of extracellular matrix in degenerated disc tissue. Eur Spine J 2005, 14: 7–26.View Article
  47. Anderson DG, Li X, Balian G: A fibronectin fragment alters the metabolism by rabbit intervertebral disc cells in vitro. Spine 2005, 30: 1242–1246. 10.1097/01.brs.0000164097.47091.4cView ArticlePubMed
  48. Xia M, Zhu Y: Fibronectin fragment activation of ERK increasing integrin a5 and b1 subunit expression to degenerate nucleus pulposus cells. J Orthop Res 2011, 29: 556–561. 10.1002/jor.21273View ArticlePubMed
  49. Aota Y, An HS, Homandberg G, Thonar EJMA, Andersson GBJ, Pichika R, et al.: Differential effects of fibronectin fragment on proteoglycan metabolism by intervertebral disc cells: A comparison with articular chondrocytes. Spine 2005, 30: 722–728. 10.1097/01.brs.0000157417.59933.dbView ArticlePubMed
  50. Pulai JI, Chen H, Im H-J, Kumar S, Hanning C, Hegde PS, et al.: NF-kB mediates the stimulation of cytokine and chemokien expression by human articular chondrocytes in response to fibronectin fragments. J Immunol 2005, 174: 5781–5788.PubMed CentralView ArticlePubMed
  51. Thompson JP, Pearce RH, Schechter MT, Adams ME, Tsang IKY, Bishop PB: Preliminary evaluation of a scheme for grading the gross morphology of the human intervertebral disc. Spine 1990, 15: 411–415. 10.1097/00007632-199005000-00012View ArticlePubMed

Copyright

© Gruber et al.; licensee BioMed Central Ltd. 2012

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.

Advertisement