Lumbar Intervertebral Disc Infection: Pathology, Prevention and … · 2007. 9. 5. · Lumbar Intervertebral Disc Infection: Pathology, Prevention and Treatment Rebecca Walters, B

Lumbar Intervertebral Disc Infection: Pathology, Prevention and Treatment

Rebecca Walters, B. Biomed. Sc.

Enrolled through the Department of Pathology, Faculty of Health Sciences, The University of Adelaide.

Research conducted at The Adelaide Centre for Spinal Research, Division of Tissue Pathology,

Institute of Medical and Veterinary Science, Adelaide.

A thesis submitted for the degree of Doctor of Philosophy by Publication in the Faculty of Health Sciences, The University of Adelaide.

March 2006

2

Table of Contents

TABLE OF CONTENTS.............................................................................................................2

ABSTRACT .............................................................................................................................3

DECLARATION .......................................................................................................................5

ACKNOWLEDGMENTS ............................................................................................................6

CHAPTER 1: INTRODUCTION .................................................................................................7 1.1 Discitis ...............................................................................................................................................................7 1.2 Symptoms, signs and investigations to diagnose iatrogenic discitis ...........................................................9 1.3 Current theories for treating discitis............................................................................................................10 1.4 Current theories for preventing discitis .......................................................................................................11 1.5 Thesis Objectives ............................................................................................................................................13 1.6 References.......................................................................................................................................................14

CHAPTER 2: EFFECTS OF INTERVERTEBRAL DISC INFECTION ON THE DEVELOPING OVINE

SPINE....................................................................................................................................22 2.1 Statement of authorship ................................................................................................................................22 2.2 Title page for published manuscript ............................................................................................................23

CHAPTER 3: THERAPEUTIC USE OF CEPHAZOLIN TO PREVENT COMPLICATIONS OF SPINE

SURGERY..............................................................................................................................35 3.1 Statement of authorship ................................................................................................................................35 3.2 Title page for published manuscript ............................................................................................................36

CHAPTER 4: PROPHYLACTIC CEPHAZOLIN TO PREVENT DISCITIS IN AN OVINE MODEL.......52 4.1 Statement of authorship ................................................................................................................................52 4.2 Title page for published manuscript ............................................................................................................53

CHAPTER 5: PREVENTING AND TREATING DISCITIS: CEPHAZOLIN PENETRATION IN OVINE

LUMBAR INTERVERTEBRAL DISC..........................................................................................69 5.1 Statement of authorship ................................................................................................................................69 5.2 Title page for published manuscript ............................................................................................................70

CHAPTER 6: PENETRATION OF CEPHAZOLIN IN HUMAN LUMBAR INTERVERTEBRAL DISC...85 6.1 Statement of authorship ................................................................................................................................85 6.2 Title page for published manuscript ............................................................................................................86

CHAPTER 7: FINAL DISCUSSION ........................................................................................ 100 7.1 Summary ...................................................................................................................................................... 100 7.2 Future Directions........................................................................................................................................ 103 7.3 Conclusion ................................................................................................................................................... 104 7.4 References.................................................................................................................................................... 105

CHAPTER 8: AMENDMENTS ............................................................................................... 107 8.1 Structure and composition of the intervertebral disc .............................................................................. 107 8.2 Comparison of the human and sheep disc ............................................................................................... 108 8.3 Dosing guidelines........................................................................................................................................ 109 8.4 Chapter Amendments ................................................................................................................................. 110 8.5 References.................................................................................................................................................... 116

3

Abstract Discitis is a potential complication of any open or percutaneous spinal procedure which

involves entry into the intervertebral disc. The infection initiates an inflammatory response

which leads to endplate rupture. Although there are variations in the severity of symptoms,

the main feature of discitis is severe back pain which is not relieved by rest. The infection

may spontaneously resolve over time although incapacitating back pain may persist for many

months. In some cases serious complications result from the spread of infection to the

adjacent vertebral bodies and over time osteomyelitis will develop with resultant bone

destruction and collapse. The prognosis for many patients with discitis is poor with continual

disabling back pain, prolonged absence from gainful employment and inability to return to

daily living activities.

Clinical and experimental evidence now supports the prophylactic use of a suitable

antibiotic to prevent discitis. In South Australia cephazolin is the antibiotic of choice to

prevent or treat discitis due to Staphylococcus spp. While cephazolin has been shown to

prevent discitis after inoculation with Staphylococcus spp. it is not universally accepted.

Uncertainty exists regarding the ability of the antibiotic to enter the disc, and if it is effective

in preventing and treating discitis. This is further complicated by the lack of suitable methods

for detecting and measuring the concentration of cephazolin in the disc.

An experimental ovine model was used to investigate (a) the natural progression of

discitis in the growing lumbar spine; (b) a technique to detect and measure the concentration

of cephazolin in the disc; (c) the effect of prophylaxis when dose and time of administration

of cephazolin was varied; (d) the effect of parenteral cephazolin after discitis was established

and (e) the influence of health and age of the disc on prophylactic and parenteral treatment

with cephazolin. In a clinical study the concentration of cephazolin was measured in

degenerate human disc tissue to determine if therapeutic concentrations were achieved.

4

The ovine studies showed that discitis had no significant effect on the development of

the growing lumbar spine after one year although infection was associated with reduced disc

area and height. Preventing discitis with cephazolin was reasonably successful, regardless of

age and health of the disc. Timing of cephazolin administration was crucial to prevent discitis

in immature animals.

A high-performance liquid chromatography technique was used to measure the

concentration of cephazolin in the disc. The greatest concentration of cephazolin in ovine

discs was achieved 15 minutes after a bolus dose of intravenous antibiotic was administered,

although detectable levels were measured for a further 2 hours. The concentration of

cephazolin was not uniform across the disc with greater concentrations in the outer disc

compared to the inner disc. Although there were measurable levels of cephazolin in these

discs, it was ineffective at treating discitis once established. In the clinical study detectable

levels of cephazolin were recovered in human discs for more than 2 hours after administering

a 1-g bolus dose. The concentration of cephazolin peaked in the human discs between 37 and

53 minutes, but in only half of the discs was the concentration of cephazolin considered

therapeutic against Staphylococcus aureus.

While discitis may spontaneously resolve over time, the infected disc does not recover

to its original form. Furthermore, parenteral cephazolin was ineffective at preventing endplate

destruction once an intradiscal inoculum was established. While this study proved cephazolin

is able to enter the disc and provide reasonable protection against infection, it appears that

discitis cannot be completely abolished. The timing of prophylaxis remains a critical factor to

achieve therapeutic concentrations of cephazolin in the disc. Due to the serious complications

that result from discitis this study supports the use of prophylactic antibiotic administered at

an optimal time before the disc is violated during any spinal procedure.

5

Declaration

This work contains no material which has been accepted for the award of any other degree or

diploma in any university or other tertiary institution. To the best of my knowledge and belief

this work contains no material previously published or written by another person, except

where due reference has been made.

I give consent for this copy of my thesis, to be made available for loan and photocopying. The

author acknowledges that copyright of published works contained within this thesis resides

with the copyright holders of those works.

Rebecca Walters

March 2006

6

Acknowledgments I gratefully acknowledge my supervisors Dr Robert Moore and Professor Robert Fraser for

their supervision, advice, support and assistance with editing throughout my candidature.

I would also like to acknowledge the support from the staff at The Adelaide Centre for

Spinal Research, Institute of Medical and Veterinary Science, where the majority of this work

was carried out.

Many thanks to the staff at the Veterinary Services Division, Infectious Diseases

Laboratories and Division of Biochemistry, Institute of Medical and Veterinary Science for

their assistance during my research.

Special thanks to Mr Ted Horgan and staff at the Royal Perth Hospital for assistance

with the biochemical assay, Dr Ian Parkinson for statistical support, Mr Lance Mickan for

providing bacterial suspensions, Professor Barrie Vernon-Roberts for assistance with editing

and for his enthusiasm for research, Dr David Hall and Dr Chris Cain for their support and

advice on clinical issues, Dr Razmi Rahmat and Dr Yoshio Shimamura for their surgical

assistance and international jokes and Dr Felicity Johnson for assistance with proof reading.

I would also like to thank the Faculty of Health Sciences, The University of Adelaide

for awarding me a Postgraduate Travelling Fellowship and DePuy Spine for awarding me the

DePuy Spinal Fellowship. I also acknowledge the support of Mayne Pharma Pty Ltd.,

Australia for supplying antibiotics for the research studies.

I am very appreciative of the funding and support I received to travel and present this

research at international and national meetings including the International Society for the

Study of the Lumbar Spine and the Spine Society of Australia.

Finally I would like to thank the Department of Pathology, The University of Adelaide

for allowing me to conduct my PhD with them and enabling me to submit my thesis by

publication.

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Chapter 1: Introduction

1.1 Discitis

Discitis, or disc space infection, is an inflammatory condition of the intervertebral disc that

was first described clinically in 1953 by Turnbull.1 The inflammatory response causes local

oedema, ischaemia, and necrosis, affecting the normal mechanical function of the disc and

surrounding tissue. The resultant back pain is often severe, and not relieved by rest.2

‘Iatrogenic’ or ‘postprocedural’ discitis occurs predominantly in adults.2 Historically

iatrogenic discitis was attributed to a chemical or aseptic reaction in the disc3-5 because

bacteria were rarely cultured. However it is now known that iatrogenic discitis can occur as a

primary infection of the disc space following an invasive surgical procedure (discectomy,

discography, percutaneous nucleotomy, laminectomies or lumbar puncture) of the spine.6,7 It

is possible, albeit rare, for children to present with iatrogenic discitis, and while spinal surgery

in this group is uncommon, inadvertent penetration of the disc during lumbar puncture can

cause iatrogenic discitis.8,9

Iatrogenic discitis occurs in up to 4% of all spinal surgeries.6,10,11 Infection is usually

due to micro-organisms originating from the patient. Alternatively, organisms may be from

the operating room environment, including theatre personnel.12 Staphylococci

(Staphylococcus aureus) and coagulase negative-staphylococci (Staphylococcus epidermidis)

are common skin organisms which cause the vast majority of infections.10 Aerobic gram-

negative bacilli and Propionibacterium species account for most of the remainder.13 Other less

common organisms include Streptococcus viridans and other Streptococcus species,

Escherichia coli, Pseudomonas aeruginosa and Mycobacterium tuberculosis and fungus.10

Organisms are inadvertently introduced into the disc space by direct implantation

through surgical instruments. The organisms flourish, eventually initiating a vascular response

and a sequence of inflammatory reactions. This leads to endplate rupture which in turn causes

8

herniation of disc material (predominantly nucleus pulposus) into the vertebral bodies. When

the endplates are breached the infecting organisms are usually cleared by the body’s immune

response. Once cleared, granulation tissue fills the void caused by the herniation and, over

time, new bone will form.14,15 In a few cases the infection may spread to the adjacent vertebral

bodies with resultant osteomyelitis causing bone destruction and collapse.15

Spontaneous primary discitis in children most commonly affects the lumbar discs of

individuals younger than 5 years of age.32-33 The condition is usually a benign and self-

limiting inflammatory reaction of the disc.19,34,35 Infecting organisms from elsewhere in the

body such as the genitourinary tract, skin or soft tissue and respiratory system enter the disc

space through haematogenous spread.34,36 Unlike adults children have a direct blood supply

to the intervertebral disc37,38 as well as numerous paravertebral and intraosseous collateral

arteries. It is rare for adults to develop spontaneous primary discitis due to regression of the

blood supply to the disc by the second decade.38

Spontaneous secondary discitis usually results from infection of the vertebral body

(osteomyelitis of the vertebral body) that spreads to the disc. The organisms most commonly

associated with spontaneous discitis are Staphylococcus aureus, Enterobacter species,

Escherichia coli, Proteus species, Pseudomonas auruginosa and Haemophilus influenzae.9,16-

19 The infection may be aggressive and destructive, involving one or more components of the

spine,20-22 with possible further complications including nerve root compression and abscess

formation in the psoas muscle.23-25 This contiguous infection is associated with the elderly

(>65 years) and those with a previous infection16,21,26 or history of IV drug use, renal disease,

diabetes and AIDS.16,27-31

Whilst both forms of discitis (iatrogenic and spontaneous) are uncommon there has

been a recent rise in the incidence of iatrogenic discitis due to the increase in invasive

procedures to diagnose or treat back pain, and improved detection with magnetic resonance

9

imaging (MRI).13 It is likely therefore that iatrogenic discitis will become more significant in

the clinical setting.

1.2 Symptoms, signs and investigations to diagnose iatrogenic discitis

The main symptoms of adults presenting with iatrogenic discitis are back pain (with any

movement of the spine) and muscle spasm,2,10,39,40 but as these same symptoms may also be

present without infection,15 treatment may be delayed due to incorrect diagnosis.17,41 The

length of time until diagnosis can range from three days to 15 months.42

In general, back pain from iatrogenic discitis becomes gradually worse over 1 to 4

weeks and is not relieved with rest. It is only poorly relieved by narcotic analgesics.15 Fever

and signs of sepsis are not usually present.2,17 Blood culture is rarely positive and needle

biopsy may not always provide a positive bacterial finding.15,17,18,43,44 This may be due to

unsatisfactory tissue sampling,45 the infection has resolved, or antibiotics were given before

the biopsy was taken.46

Laboratory investigations include non-specific blood markers (elevated ESR and

serum CRP)41,47 and imaging (CT and MRI).10 Plain radiographs are not sufficiently sensitive

to detect the early endplate changes which may themselves, take several weeks to become

evident.41,48 Bone scans may detect osteomyelitis earlier than radiographs21 which are more

informative for long-term changes, such as bony sclerosis, reduced disc height and fusion.

MRI and CT are more sensitive and specific detecting changes in the disc space and adjacent

bone marrow that are consistent with early discitis.2,49-51

Follow-up studies have shown various outcomes after discitis. These range from a

lack of symptoms plus unrestricted spinal mobility, to destruction of adjacent vertebral

bodies, abscess formation, local kyphosis, recurrent backache, narrowing of the intervertebral

10

disc space and fusion of the vertebrae.18,52-54 Generally long-term morbidity is high with over

50% of patients unable to return to work.11,39

The detrimental outcomes and major costs of iatrogenic discitis to the patient and the

health system are due to prolonged hospitalisation, further surgery (debridement of necrotic

tissue, draining an abscess or fusion) and increased patient care. The patient suffers further

with the time lost from gainful employment,53 while potential medico-legal actions are

detrimental to the hospital or surgical staff. 12

In summary, symptoms and signs of discitis may be over looked unless the clinician

suspects infection. MRI appears to be the most sensitive tool to detect discitis early. This is

imperative as delay in diagnosis and treatment may lead to complications, increased length of

hospital stay, and further cost to the patient.

1.3 Current theories for treating discitis

Generally adults with discitis are initially treated conservatively with analgesics, antibiotics,

bed rest or immobilisation.10,39 Most authors advocate the use of antibiotics following

isolation of the organism involved, but if this is not successful, broad-spectrum antibiotics are

recommended.

The role of antibiotics in the later stages of the disease is controversial. The infection

may have concluded before diagnosis is made. As a result, general antibiotics are usually

administered to prevent subsequent infection without specific knowledge of the pathogen.40,55

Furthermore, antibiotics may be ineffective due to resistance of the causative micro-

organism.42,55

On the basis of clinical experience, some authors have questioned treatment regimens

and the efficacy of antibiotics for this condition.10,39,56 There is general agreement that a

minimum of four weeks of intravenous therapy should be given57 with some authors

11

recommending six to eight weeks.2,18 Oral antibiotics are recommended following intravenous

therapy to reduce the risk of infection relapse. 18

If conservative treatment fails further surgery may be required to treat complications

such as epidural abscess, debridement of necrotic tissue from the infected area or interbody

fusion with bone graft may be indicated.18,39,42,58,59 In one study patients with disabling back

pain from discitis who were treated surgically had a better clinical outcome than those treated

with antibiotics alone. 18

1.4 Current theories for preventing discitis

Due to the unfavourable outcome of postoperative discitis, prophylactic antibiotics are

recommended during any open or percutaneous spinal procedure that involves the disc.

Together with good aseptic surgical technique, prophylactic antibiotics have been reported to

reduce the incidence of iatrogenic discitis, as well as the incidence of post-operative wound

infections.11,15,46,60-64

For a prophylactic antibiotic to be effective it must be present in sufficient

concentration in vulnerable tissue from the time of surgical incision and for the duration of

the procedure. 65-67 In addition it needs to prevent the development of postprocedural discitis

without the risk of developing resistance and subsequent superinfection. An antibiotic used

commonly in South Australia for this purpose is cephazolin.

Cephazolin is given prophylactically as a 1-2 g dose (depending on patient weight) 30

minutes to one hour before spinal surgery and generally repeated every four hours at half the

initial dose for prolonged procedures (or following haemorrhage).13,68-70

Although cephazolin penetrates vascular tissue well, there is disagreement in the

literature regarding its ability to enter the disc in an active form.71-75 In fact the use of

prophylactic antibiotic altogether is not universally accepted.76

12

Because the healthy adult disc has no direct blood supply, nourishment occurs by

diffusion through the cartilage endplates or, up until the fourth decade, by vessels in the outer

annulus fibrosus.38 As is the case for all antibiotics administered intravenously, cephazolin

must diffuse through the capillary beds of the cartilage endplates to enter the adult disc. The

ability of the antibiotic to diffuse through all parts of the disc may be influenced by the

structure of the disc (vascular supply, size and health) and properties of the drug (size,

solubility, binding and charge). Particularly, charge of the antibiotic has been discussed in the

literature.

The nucleus pulposus is rich in glycosaminoglycans and has a high density of negative

charge. It has been postulated that positively charged antibiotics (gentamicin and

vancomycin) can enter the disc, whereas negatively charged antibiotics (penicillin and

cephazolin) have limited46,65,77 or no78 penetration because of mutually repellent charges.71-75

It is questionable whether cephazolin reaches therapeutic levels in all regions of the

disc. Few studies have measured and reported actual levels of cephazolin in the human

spine.65,77 Although methods for detecting cephazolin are well documented,79-90 not many are

applicable to the avascular intervertebral disc. Most papers describe indirect methods of

measuring antibiotic concentration.46,63,65,78

If cephazolin could not enter the disc it would fail as a prophylactic. However,

cephazolin has been shown to prevent discitis after inoculation with Staphylococcus spp. in

animal models.46,61,91 These results suggest if cephazolin is administered at an appropriate

time and dose it can be successful at preventing discitis. In spite of this, conclusive studies to

prove that the antibiotic can enter the disc, and is effective as a prophylaxis, are currently not

evident in the literature. In fact, despite a better general understanding of the pathogenesis of

disc infection,46,61 little is known about the ability of any antibiotics to penetrate the disc.46,75

13

1.5 Thesis Objectives To resolve the diversity of opinion about strategies for the treatment and prevention of

discitis, and on the choice, dose and timing of antibiotic administration, this thesis aims to

determine:

1. Whether discitis without antibiotic prophylaxis or treatment influences development of the

immature sheep spine.

2. The concentration of cephazolin in nucleus pulposus and annulus fibrosus tissue of the

ovine and human disc after administration of a bolus dose.

3. Whether cephazolin given at intervals over a four-hour period can prevent iatrogenic

discitis in immature and mature ovine discs.

4. If disc degeneration in the sheep influences the tissue levels of cephazolin and its

effectiveness in preventing or treating discitis.

5. If the concentration of cephazolin in human disc and blood reach the minimum inhibitory

concentration (MIC) specific for Staphylococcus aureus.

14

1.6 References

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17

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4.

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35.

63. Osti OL, Fraser RD, Vernon-Roberts B. Discitis after discography. The role of

prophylactic antibiotics. J Bone Joint Surg Br 1990;72:271-4.

64. Schnoring M, Brock M. Prophylactic antibiotics in lumbar disc surgery: analysis of

1,030 procedures. [in German] Zentralbl Neurochir 2003;64:24-9.

65. Boscardin JB, Ringus JC, Feingold DJ, et al. Human intradiscal levels with cefazolin.

Spine 1992;17:S145-8.

66. Luer MS, Hatton J. Appropriateness of antibiotic selection and use in laminectomy

and microdiskectomy. Am J Hosp Pharm 1993;50:667-70.

67. Lang R, Folman Y, Ravid M, et al. Penetration of ceftriaxone into the intervertebral

disc. J Bone Joint Surg Am 1994;76:689-91.

68. Bennett JV, Brachman PS eds. Hospital infections. 4th ed. Philadelphia: Lippincott-

Raven, 1998.

69. Dimick JB, Pronovost PJ, Lipsett PA. Spine update: antimicrobial prophylaxis in spine

surgery: basic principles and recent advances. Intensive Care Med 2000;26:1857-62.

70. Swoboda SM, Merz C, Kostuik J, et al. Does intraoperative blood loss affect antibiotic

serum and tissue concentrations? Arch Surg 1996;131:1165-71.

20

71. Currier BL, Banovac K, Eismont FJ. Gentamicin penetration into normal rabbit

nucleus pulposus. Spine 1994;19:2614-8.

72. Eismont FJ, Wiesel SW, Brighton CT, et al. Antibiotic penetration into rabbit nucleus

pulposus. Spine 1987;12:254-6.

73. Thomas Rde W, Batten JJ, Want S, et al. A new in-vitro model to investigate

antibiotic penetration of the intervertebral disc. J Bone Joint Surg Br 1995;77:967-70.

74. Scuderi GJ, Greenberg SS, Banovac K, et al. Penetration of glycopeptide antibiotics in

nucleus pulposus. Spine 1993;18:2039-42.

75. Gibson MJ, Karpinski MR, Slack RC, et al. The penetration of antibiotics into the

normal intervertebral disc. J Bone Joint Surg Br 1987;69:784-6.

76. Willems PC, Jacobs W, Duinkerke ES, et al. Lumbar discography: should we use

prophylactic antibiotics? A study of 435 consecutive discograms and a systematic

review of the literature. J Spinal Disord Tech 2004;17:243-7.

77. Rhoten RL, Murphy MA, Kalfas IH, et al. Antibiotic penetration into cervical discs.

Neurosurgery 1995;37:418-21.

78. Riley L, Banovac K, Martinez O, et al. Tissue distribution of antibiotics in the

intervertebral disc. Spine 1994;19:2619-25.

79. Beovic B, Mrhar A, Karba R, et al. Influence of fever on cefazolin pharmacokinetics.

J Chemother 1999;11:40-45.

80. Galanti LM, Hecq JD, Vanbeckbergen D, et al. Long-term stability of cefuroxime and

cefazolin sodium in intravenous infusions. J Clin Pharm Ther1996;21:185-9.

81. Gupta VD, and Stewart KR. Quantitation of carbenicillin disodium, cefazolin sodium,

cephalothin sodium, nafcillin sodium, and ticarcillin disodium by high-pressure liquid

chromatography. J Pharm Sci 1980;69:1264-7.

82. How TH, Loo WY, Yow KL, et al. Stability of cefazolin sodium eye drops. J Clin

Pharm Ther 1998;23:41-7.

21

83. Hume AL, Polk R, Kline B, et al. Comparative penetration of latamoxef (moxalactam)

and cefazolin into human knee following simultaneous administration. J Antimicrob

Chemother 1983;12:623-7.

84. Lanao JM, Vicente MT, Dominguez-Gil A. Pharmacokinetics of cefazolin

administered as a new drug delivery system in healthy volunteers. Biopharm Drug

Dispos 1988;9:377-88.

85. Liang D, Chow D, White C. High-performance liquid chromatographic assay of

cefazolin in rat tissues. J Chromatogr B Biomed Appl 1994;656:460-5.

86. Low CL, Gopalakrishna K, Lye WC. Pharmacokinetics of once daily intraperitoneal

cefazolin in continuous ambulatory peritoneal dialysis patients. J Am Soc Nephrol

2000;11:1117-21.

87. Polk R, Hume A, Kline BJ et al. Penetration of moxalactam and cefazolin into bone

following simultaneous bolus or infusion Clin Orthop 1983:177:216-21.

88. Walker, PC, Kaufmann RE, Massoud N. Compatibility of cefazolin and gentamicin in

peritoneal dialysis solutions. Drug Intell Clin Pharm 1986;20:697-700.

89. Watanabe Y, Hayashi T, Takada R, et al. Studies on protein binding of antibiotics. Ι.

Effect of cefazolin on protein binding and pharmacokinetics of cefoperazone. J

Antibiot (Toyko) 1980;33:625-35.

90. Wold JS and Turnipseed SA. The simultaneous quantitative determination of

cephalothin and cefazolin in serum by high pressure liquid chromatography. Clin

Chim Acta 1977;78:203-7.

91. Osti OL, Vernon-Roberts B, Fraser RD. 1990 Volvo Award in experimental studies.

Anulus tears and intervertebral disc degeneration. An experimental study using an

animal model. Spine 1990;15:762-7.

Chapter 2: Effects of intervertebral disc infection on the developing ovine spine

2.1 Statement of authorship

EFFECTS OF INTERVERTEBRAL DISC INFECTION ON THE DEVELOPING

OVINE SPINE

SPINE 2005;30:1252-1257. WALTERS, R.M. (Candidate) Assisted surgery, prepared and performed analysis on samples, interpreted data, contributed to manuscript writing. Signed……………………………………………………..Date………………….. SMITH, S.H.E. Performed analysis of the data and contributed to manuscript writing. Signed……………………………………………………..Date………………….. HUTCHINSON, J.H. Performed surgery. Signed……………………………………………………..Date………………….. DOLAN, A.M. Performed surgery. Signed……………………………………………………..Date………………….. FRASER, R.D. Performed surgery, supervised development of work, helped in data interpretation and manuscript evaluation. Signed……………………………………………………..Date………………….. MOORE, R.J. Supervised development of work, helped in data interpretation, contributed to manuscript writing and evaluation, acted as corresponding author. Signed……………………………………………………..Date…………………..

23

2.2 Title page for published manuscript

CHAPTER 2

EFFECTS OF INTERVERTEBRAL DISC INFECTION ON THE DEVELOPING OVINE SPINE

R.M. Walters1, 2, S.H.E Smith1, M.J. Hutchinson3, A.M. Dolan3, R.D. Fraser1, 3, R.J. Moore1, 2

1The Adelaide Centre for Spinal Research, Institute of Medical and Veterinary Science. 2Department of Pathology, The University of Adelaide

3The Spinal Unit, Royal Adelaide Hospital, Adelaide, Australia

SPINE 2005;30:1252-1257.

Walters, R.M., Smith, S.H.E., Hutchinson, M.J., Dolan, A.M., Fraser, R.D. & Moore, R.J. (2005) Effects of intervertebral disc infection on the developing ovine spine. Spine v.30 (11) pp. 1252-1257, June 1 2005

NOTE: This publication is included on pages 23 – 34 in the print copy of the thesis held in the University of Adelaide Library.

35

Chapter 3: Therapeutic use of cephazolin to prevent complications of spine

surgery


THERAPEUTIC USE OF CEPHAZOLIN TO PREVENT COMPLICATIONS OF

SPINE SURGERY

INFLAMMOPHARMACOLOGY 2006;14:138-143

WALTERS, R.M. (Candidate)

Collected samples, prepared samples for analysis, interpreted data, contributed to manuscript writing and acted as corresponding author. Signed……………………………………………………..Date…………………..

VERNON-ROBERTS, B

Supervised development of work and manuscript evaluation.

Signed……………………………………………………..Date………………….. FRASER, R.D. Supervised development of work and manuscript evaluation. Signed……………………………………………………..Date………………….. MOORE, R.J. Supervised development of work and manuscript evaluation. Signed……………………………………………………..Date…………………..

36


CHAPTER 3

THERAPEUTIC USE OF CEPHAZOLIN TO PREVENT COMPLICATIONS OF

SPINE SURGERY

R.M. Walters,1, 2 B. Vernon-Roberts,1 R.D. Fraser,3 R.J. Moore 1, 2

1The Adelaide Centre for Spinal Research, Institute of Medical and Veterinary Science

2Department of Pathology, The University of Adelaide

3 Spinal Unit, Royal Adelaide Hospital, Adelaide, South Australia

INFLAMMOPHARMACOLOGY 2006;14:138-143

Walters, R.M., Vernon-Roberts, B., Fraser, R.D. & Moore, R.J. (2006) Therapeutic use of cephazolin to prevent complications of spine surgery. Inflammopharmacology v.14 (3/4) pp. 138-143, August 2006

NOTE: This publication is included on pages 36 - 51 in the print

copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1007/s10787-006-1503-y

http://dx.doi.org/10.1007/s10787-006-1503-y

52

Chapter 4: Prophylactic cephazolin to prevent discitis in an ovine model


PROPHYLACTIC CEPHAZOLIN TO PREVENT DISCITIS IN AN OVINE MODEL

SPINE 2006;31:391-396.

WALTERS, R.M. (Candidate)

Assisted surgery, prepared and performed analysis on samples, interpreted data, contributed to

manuscript writing and acted as corresponding author.

Signed……………………………………………………..Date…………………..

RAHMAT, R.

Performed surgery.

Signed……………………………………………………..Date…………………..

SHIMAMURA, Y.

Performed surgery.

Signed……………………………………………………..Date…………………..

FRASER, R.D.

Performed surgery, supervised development of work, helped in data interpretation and

manuscript evaluation.

Signed……………………………………………………..Date…………………..

MOORE, R.J.

Supervised development of work, helped in data interpretation, contributed to manuscript

writing and evaluation.

Signed……………………………………………………..Date…………………..

53


CHAPTER 4

PROPHYLACTIC CEPHAZOLIN TO PREVENT DISCITIS IN AN OVINE MODEL

R.M. Walters1, 2, R. Rahmat1, Y. Shimamura1, R.D. Fraser1, 3, R.J. Moore1, 2

1The Adelaide Centre for Spinal Research, Institute of Medical and Veterinary Science.

2Department of Pathology, The University of Adelaide


SPINE 2006;31:391-396.

Walters, R.M., Rahmat, R., Shimamura, Y., Fraser, R.D. & Moore, R.J. (2006) Prophylactic cephazolin to prevent discitis in an ovine model. Spine v.31 (4) pp. 391-396, February 15 2006


69

Chapter 5: Preventing and treating discitis: Cephazolin penetration in ovine lumbar intervertebral disc


PREVENTING AND TREATING DISCITIS: CEPHAZOLIN PENETRATION IN

OVINE LUMBAR INTERVERTEBRAL DISC

EUROPEAN SPINE JOURNAL (Submitted)

WALTERS, R.M. (Candidate) Assisted surgery, prepared and performed analysis on samples, interpreted data, contributed to manuscript writing and acted as corresponding author. Signed……………………………………………………..Date………………….. RAHMAT, R. Performed surgery. Signed……………………………………………………..Date………………….. FRASER, R.D. Performed surgery, supervised development of work, helped in data interpretation and manuscript evaluation. Signed……………………………………………………..Date………………….. MOORE, R.J. Supervised development of work, helped in data interpretation, contributed to manuscript writing and evaluation. Signed……………………………………………………..Date…………………..

70


CHAPTER 5

PREVENTING AND TREATING DISCITIS: CEPHAZOLIN PENETRATION IN OVINE LUMBAR INTERVERTEBRAL DISC

R.M. Walters1, 2, R. Rahmat1, R.D. Fraser1, 3, R.J. Moore1, 2



EUROPEAN SPINE JOURNAL (Submitted)

Walters, R.M., Rahmat, R.,Fraser, R.D. & Moore, R.J. (2006) Preventing and treating discitis: cephazolin penetration in ovine lumbar intervertebral disc. European Spine Journal v.15 (9) pp. 1397-1403, September 2006

NOTE: This publication is included on pages 70 - 84 in the print

copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1007/s00586-006-0144-6

http://dx.doi.org/10.1007/s00586-006-0144-6

85

Chapter 6: Penetration of cephazolin in human lumbar intervertebral disc


PENETRATION OF CEPHAZOLIN IN HUMAN LUMBAR INTERVERTEBRAL DISC

SPINE 2006;31:567-570.

WALTERS, R.M. (Candidate) Prepared and performed analysis on samples, interpreted data, contributed to manuscript writing and acted as corresponding author. Signed……………………………………………………..Date………………….. FRASER, R.D. Performed surgery, supervised development of work, helped in data interpretation and manuscript evaluation. Signed……………………………………………………..Date………………….. MOORE, R.J. Supervised development of work, helped in data interpretation, contributed to manuscript writing and evaluation. Signed……………………………………………………..Date…………………..

86


Chapter 6

PENETRATION OF CEPHAZOLIN IN HUMAN LUMBAR INTERVERTEBRAL DISC

R.M. Walters1, 2, R.D. Fraser1, 3, R.J. Moore1, 2



SPINE 2006;31:567-570.

Walters, R.M., Fraser, R.D. & Moore, R.J. (2006) Penetration of cephazolin in human lumbar intervertebral disc. Spine v.31 (5) pp. 567-570, March 1 2006


100

Chapter 7: Final Discussion

7.1 Summary

With Animal and Human Ethics Committee approval, research proposals were conducted to

resolve the diversity of opinion regarding the long-term effects of discitis on the growing

spine, and the effectiveness of cephazolin to prevent and treat iatrogenic discitis. The

significant findings from these studies are summarised.

Injecting ovine discs with Staphylococcus spp. initiated vascular and inflammatory

reactions that led to changes typically associated with discitis. Infection with Staphylococcus

aureus and Staphylococcus epidermidis initiated endplate rupture which in turn caused

herniation of disc material. S. aureus produced a more aggressive response than S.

epidermidis. The lesions from S. aureus infection were haemorrhagic, irregular, extended well

into the adjacent vertebral body, and almost always changed in size and shape over time.

Lesions from S. epidermidis infection were less invasive, with minimal erosion of the

vertebral endplate and remained relatively unchanged in size and shape over 12 weeks which

may explain that while inoculation of S. epidermidis in immature ovine discs results in

retardation of disc development, it had no significant effect on vertebral body growth or

growth of the entire lumbar spine. These results concur with long-term studies that discitis in

children is relatively benign and the potential for bone destruction is low.1

Spontaneous discitis in children is generally asymptomatic and typically has a benign

outcome. Some authors also believe long-term outcome of childhood discitis is not dependent

on treatment with antibiotics.2-4 This may be attributed to the healing capacity of the disc due

to the abundant vascularity present at that age. An increased number of vascular channels in

the cartilaginous endplate and disc may promote clearance of bacteria while limiting tissue

damage. It was most probable that the threefold increase in blood vessels occupying the

101

endplate in sheep less than 6 months old assisted the clearance of the inoculum, because only

a half of all inoculated discs developed discitis.

In adult iatrogenic discitis bacteria are not cleared by an immune response until the

endplates are breached and blood vessels are prevalent. To prevent infection and the

subsequent inflammatory response destroying the surrounding tissue, prophylactic antibiotics

must enter the disc. Cephazolin, like all molecules, can only enter the disc by passive

diffusion.5 The distribution of the antibiotic in the disc is controlled by the high concentration

of glycosaminoglycan molecules in the nucleus pulposus which creates a high density of

relative negative charge.6 As a result positively charged molecules are more likely to enter the

disc from the capillary bed of the endplate and negatively charged molecules predominantly

enter through the outer annulus.7 Cephazolin is a weak acid with a low pKa and an overall

negative charge. Theoretically the route of entry of unbound cephazolin should be through the

meagre supply of vessels in the outer annulus fibrosus but due to its negative charge high

concentrations of cephazolin within the nucleus pulposus would not be expected.

The results from the high-performance liquid chromatography assay confirmed

detectable levels of cephazolin in all regions of the ovine disc. Although the distribution was

not uniform, it was nonetheless promising that it could be detected as early as 15 minutes

after intravenous administration. Consistent with the theory that relative negative charge plays

a significant role in molecular distribution there was higher concentrations of cephazolin in

the annulus fibrosus compared to the nucleus pulposus. In fact, increasing the intravenous

dose of cephazolin from 2 g to 4 g did not significantly increase the concentration of

cephazolin in the nucleus pulposus, further supporting the charge theory.

By incising the outer annulus fibrosus and partially removing the nucleus pulposus

(consisting of negatively charged glycosaminoglycan molecules) it was hypothesised that

vascularity of the disc would increase and relative negative charge of the disc would decrease

and therefore facilitate the penetration of antibiotic into the disc and reduce the incidence of

102

discitis. Despite histologic confirmation of vascular granulation tissue in the outer annulus

fibrosus and partial nucleotomy, the concentration of cephazolin was not significantly

different compared to non-incised discs. It would appear that this treatment had no significant

effect on the delivery of antibiotics to the ovine disc and did not influence the incidence of

discitis. However, altering the timing of administration of cephazolin significantly influenced

the incidence of discitis in immature sheep. Although the immature discs had an abundant

vascular supply and higher concentration of cephazolin, discitis developed 10 times more

readily in lambs than in mature sheep. However, prophylactic cephazolin administered 30

minutes before inoculation consistently prevented discitis in both mature and immature sheep.

While the ovine model was able to replicate a human disease process and suggest what

is likely to occur in the human spine, caution is required when extrapolating data to humans.

Although the structure and composition of the sheep disc is remarkably similar to the human

disc there are differences. Mature sheep discs are approximately half the size of human discs

and contain a growth plate, whereas the human disc has a ring epiphysis. It would be

reasonable to assume that cephazolin would take longer to diffuse through a larger disc, but

other factors such as health, weight and gender may confound the interpretation of the results.

After administering cephazolin (IV) to humans, the concentration of cephazolin was

greatest in samples of discs collected between 37 and 53 minutes. Importantly therapeutic

concentrations (against Staphylococcus aureus) were not detected in all samples over the 2

hour period and the period of detection varied considerably between individuals. Along with

external factors, this variability may have been attributed to differences in vascular supply,

size and maturity of the disc and may explain the differences in timing of prophylaxis

between the ovine and human disc.

103

7.2 Future Directions Intravenous administration of cephazolin was not the most efficient method of delivery to the

disc, particularly to the nucleus pulposus. Further studies are required to determine if

intradiscal injection, in combination with intravenous administration, would be a better

option. Intradiscal injection would facilitate direct delivery of the antibiotic into the disc and

potentially distribute it evenly throughout the disc. It would also overcome problems such as

drug-binding, size exclusion and poor vascular supply. Intradiscal injections may benefit the

patient during open or percutaneous procedures, but would not provide systemic prophylaxis

or be appropriate for long-term antibiotic treatment of discitis.

Analytical methods that complement microbiological culture methods may provide a

better understanding of the types of organisms that cause infection. DNA-based methods8,9

that identify the bacterial DNA causing discitis may become more significant. These methods

are highly sensitive and specific and can identify bacterial species in patients with negative

blood and disc aspirate cultures. Accurate and early diagnosis will enable organism specific

antibiotics to be administered early. This may prevent endplate erosion, and the further

complications of discitis such as osteomyelitis or abscess formation.

Further research involving the detection of antibiotic in the disc is required. The

method described in this thesis, to detect the concentration of cephazolin in the disc, provided

only one measurement at a single time point. It would have been more appropriate and

accurate if multiple measurements of antibiotic concentration were taken from one disc.

Potentially, multiple measurements of antibiotic concentration could be taken throughout a

surgical procedure while the patient is undergoing surgery. Although such methods have not

been established for the disc, tissue microdialysis and capillary electrophoresis have been

used to determine antibiotic concentration in organs and fluid of the body.10-12 These

applications are designed to take serial samples over time and provide an opportunity to

quantify tissue drug distribution in vivo. This would show the actual rise and decline of the

104

concentration of the antibiotic in the disc and would improve our understanding of the factors

that influence antibiotic concentration, the individual variation that occurs across a population

and the effects on the disc.

7.3 Conclusion It is clear from these studies that iatrogenic discitis is detrimental to the disc and prophylactic

antibiotics are necessary to prevent the outcome of infection. Intravenous cephazolin is a

reasonable choice as a prophylactic antibiotic. Cephazolin could be detected in ovine and

human disc however it was unable to penetrate all aspects of the disc uniformly. The uneven

distribution of cephazolin in the disc may influence the incidence of discitis, as infection

could not be completely abolished over a period of time. Timing of prophylactic

administration remains critical to provide the best protection to the disc during open or

percutaneous procedures.

105

7.4 References 1. Crawford AH, Kucharzyk DW, Ruda R, et al. Diskitis in children. Clin Orthop

1991;266:70-9.

2. Ventura N, Gonzalez E, Terricabras L, et al. Intervertebral discitis in children: a

review of 12 cases. Int Orthop 1996;20:32-4.

3. Jansen BR, Hart W, Schreuder O. Discitis in childhood. 12-35-year follow-up of 35

patients. Acta Orthop Scand 1993;64:33-6.

4. Spiegel PG, Kengla KW, Isaacson AS, et al. Intervertebral disc-space inflammation in

children. J Bone Joint Surg Am 1972;54:284-96.

5. Rudert M and Tillmann B. Detection of lymph and blood vessels in the human

intervertebral disc by histochemical and immunohistochemical methods. Ann Anat

1993;175:237-42.

6. Urban J. Disc Biochemistry in Relation to Function. second ed. Philadelphia: W.B.

Saunders Company, 1996.

7. Holm S, Maroudas A, Urban JP, et al. Nutrition of the intervertebral disc: solute

transport and metabolism. Connect Tissue Res 1981;8:101-19.

8. Fritzell P, Bergstrom T, Welinder-Olsson C. Detection of bacterial DNA in painful

degenerated spinal discs in patients without signs of clinical infection. Eur Spine J

2004;13:702-6.

9. Lecouvet F, Irenge L, Vandercam B, et al. The etiologic diagnosis of infectious

discitis is improved by amplification-based DNA analysis. Arthritis Rheum

2004;50:2985-94.

10. Klekner A, Ga'spa'r A, Kardos S, et al. Cefazolin prophylaxis in neurosurgery

monitored by capillary electrophoresis. J Neurosurg Anesthesiol 2003;15:249-54.

11. Joukhadar C, Derendorf, H, Muller M. Microdialysis. A novel tool for clinical studies

of anti-infective agents. Eur J Clin Pharmacol 2001;57:211-9.

106

12. Lorentzen H, Kallehave F, Kolmos HJ, et al. Gentamicin concentrations in human

subcutaneous tissue. Antimicrob Agents Chemother 1996;40:1785-9.

107

Chapter 8: Amendments

8.1 Structure and composition of the intervertebral disc The intervertebral discs are complex cartilaginous structures located between the vertebral

bodies which allow the otherwise rigid spine to move in flexion, extension and rotation. The

disc is comprised of a central nucleus pulposus enclosed by concentric layers of collagen

which make up the annulus fibrosus. Located superiorly and inferiorly from the intervertebral

disc are the cartilage endplates, adjacent to the vertebral bodies.

The major components of the disc are water, fibrillar collagen and aggrecan

(proteoglycans consisting of glycosaminoglycan chains). However from birth the ratio and

composition of these components in the disc changes.1 The most significant changes occur

from birth up to the second decade of life and again with advancing age (> 40 years).2

At birth the nucleus pulposus comprises half of the disc space. It is clear, gelatinous

and highly hydrated with a high concentration of aggrecan but a relatively lower collagen

content.3 The large aggregating proteoglycans consist of a protein core and sulphated

glycosaminoglycan (GAG) chains. These side chains which have a high density of negative

charges associated with them, are responsible for the distribution of molecules throughout the

disc.3 Surrounding the nucleus pulposus are the abundant and firm collagen fibres of the

annulus fibrosus that form concentric rings or lamellae. The lamellae of the immature annulus

fibrosus contain blood vessels, which supply some nutrients to the disc. The collagen fibres of

the annulus fibrosus continue to extend laterally to form the hyaline cartilage of the

epiphyseal end plates. In the immature spine this functions as the growth plate for the adjacent

vertebral body, containing a large blood supply that provides the majority of nutrition to the

intervertebral disc.3

With maturity, the collagen fibres of the annulus fibrosus thicken and the proportion

of aggrecan and water in the nucleus pulposus decreases.3,4 The structure of the cartilage

108

endplate changes to form a layer of hyaline cartilage which calcifies and joins the bone,

forming the ring epiphysis.5 By the second decade of life blood vessels in the annulus fibrosus

and endplates have regressed, reducing the nutrient supply to the disc.6,7 Until the fourth

decade of life there is no direct blood supply to the healthy adult disc.6 Nutrient delivery to the

centre of the disc is predominantly by passive diffusion across the endplates.7

Progressive changes continue to occur later in life. These include a loss of disc height8

an increase in number of clefts and tears of the nucleus pulposus, neovascularisation of the

outer annulus and endplate,9 disorganization, calcification and thinning of the endplate, and

cellular changes leading to degenerative disc disease.2

8.2 Comparison of the human and sheep disc Although the sheep is a quadruped, the structure and composition of sheep discs is

remarkably similar to human discs.10 Sheep discs also consist of an inner nucleus pulposus

and outer annulus fibrosus containing water, fibrillar collagen and aggrecan that change in

proportion throughout life. The most notable difference between the two species is size. The

anterior disc height in the mature sheep lumbar spine is approximately 5mm lower than in

humans.11 Likewise the size of the vertebral bodies is also notably different. Human vertebral

bodies are wider than tall and contain a ring epiphysis compared to the sheep vertebral bodies

which are taller than wide and contain a growth plate. However both species have a

pronounced oval shaped vertebral body.10

Immature lamb discs have a highly vascular cartilage endplate that supplies nutrients

to the disc. However, by 6 months of age the blood vessels regress, resulting in a mature

avascular disc. Similar to the human disc, the mature sheep relies on diffusion of nutrients

through the capillary bed.

109

The sheep is a valid model for human spinal conditions and has been utilised over two

decades to demonstrate intervertebral disc and vertebral pathology of the spine.11-14 Sheep are

readily available in Australia, reasonably inexpensive and show much more homogeneity than

do human specimens when selected for breed, sex, age and weight.15 Its spinal biomechanics

are similar to the human spine and it is large enough and a useful model for a range of

surgical procedures.10

While the sheep model is useful to understand the pathology of disease and the likely

consequences in the human, one is mindful of the need to be cautious when extrapolating data

from an animal to recommend clinical decisions for the human. Despite such variations,

patients will ultimately benefit from improved understanding and clinical practice derived

from knowledge gained from animal models. In summary, there are proven sheep models for

discitis and disc degeneration and these are used in the experimental chapters described in this

thesis.

8.3 Dosing guidelines An introduction to dosing guidelines of cephazolin are described on pages 10 and 11 of the

thesis and more detailed information is provided in the methods section of each paper

respectively. However, a brief description of dosing guidelines is outlined below.

Prophylactic antibiotics are given as either a single intravenous dose at induction of

anaesthesia or in combination with an intradiscal dose during surgery. Cephazolin is

administered prophylactically as a 1-2 g dose 30 minutes to one hour before surgery and

generally repeated (every four hours) at half the initial dose for prolonged procedures or

following haemorrhage. Continuing antibiotics for longer than 24 hours is not advisable as

this may promote resistant microbial pathogens, expose the patient to more adverse drug

effects and increase medical costs.

110

Cephazolin is also indicated in the treatment of bone and joint infections due to S.

aureus. In adults the usual dose for moderate to severe infections is 500 mg to 1 g every six to

eight hours. In children a total daily dosage of 25 to 50 mg per kg of body weight, divided

into three or four equal doses is effective for most infections (Mayne Pharma Pty Ltd.,

Australia).

8.4 Chapter Amendments

8.4.1 Chapter 2 page 27, paragraph 2

American Type Culture Collection (ATCC) is a collection of bacterial cultures used as quality

control organisms for research purposes to identify the culture type used.


Previous work using the animal model of discitis in the mature sheep with S. epidermidis as

the inoculum has been reported but none have looked at the long-term effects of this organism

in the immature sheep spine. Although S. aureus is a common infecting organism,

Staphylococcus species (including S. epidermidis) and other gram-negative bacteria are also

implicated.


The Sagittal Convexity Index is the central disc height (D), measured as the maximal

thickness of the disc is divided by the sum of the anterior (E) and posterior disc heights (F),

taken from the peripheral endplates.

111


The Farfan Index is a measure of the sum of the anterior and posterior disc height as a

percentage of the width of the vertebral body.


The histological features of new bone formation were identified using polarised light

microscopy. The collagen fibres were typically arranged in an irregular form suggesting

formation of new bone (woven bone).


Chronic inflammation is due to microorganisms that are able to resist phagocytosis and incite

an inflammatory response, which results in significant tissue damage. Significant tissue

damage can be seen within and surrounding the intervertebral disc. Eosinophilic staining scar

tissue shown in the low power view is evidence of a chronic inflammatory response. Scar

tissue has developed at the site of the lesion, most likely in response to the release of

cytokines by the activated macrophages (not shown).


Although chronic inflammatory cells (activated macrophages and lymphocytes) cannot be

distinguished from one another in a low power view, in a high power view a cellular response

is evident. An influx of polymorphonuclear cells surrounds the lesion, between the cartilage

endplate and epiphyseal growth plate. Cellular detail is not shown in Figure 8, as this is a low

power view to visualise the extent of the lesion.

112


The regression line is a straight line that passes through the origin (zero) and the values of the

working (stock) standards (0, 16, 80 and 400 mg/L for plasma and 0.64, 3.2, 16 mg/L for disc

tissue) to determine the accuracy of the assay using sheep plasma and disc tissue. The

accuracy of the standards is determined by the deviation of actual values compared to the

regression line.


The difference in tissue cephazolin concentration may be related to the extraction method

because of the time taken to remove disc tissue from the sheep spine or possibly the disc level

and variability in disc size.


One must be mindful that this is a method paper to describe the technique and not a research

paper to understand the mechanisms responsible for antibiotic penetration into the different

regions of the disc. The number of sheep in this study was two. Determining statistics for the

concentration of cephazolin would not be appropriate or accurate. Sample results described in

this paper are reported as evidence that the technique is suitable for sheep samples. The

validity of this method is confirmed by statistics completed for the extraction technique on

ovine disc tissue and plasma standards for every assay run (standard curve) and recovery.


The nucleus pulposus has a high concentration of proteoglycans which produce a high-density

negative charge. Cephazolin is a weak acid with a low pKa and negative charge. It is assumed

the high concentration of proteoglycans in the nucleus pulposus tissue controls the

distribution of cephazolin in the disc. The diffusion of cephazolin into the central region of

113

the disc is restricted by the repulsion of the like charges. Therefore concentration of

cephazolin in the outer disc (annulus pulposus) remains greater.


Discs that were infected and developed discitis were not used to determine concentration of

cephazolin in the disc. Discitis causes vascular ingrowth which significantly influences the

cephazolin concentration in the disc. Discitis also makes it difficult to identify the structural

components of the disc thereby making it difficult to accurately separate annulus fibrosus

tissue from nucleus pulposus tissue.


Although incision of the annulus fibrosus initiates a highly vascular granulation tissue

response in the peripheral layers, this does not appear to significantly influence the diffusion

of antibiotic into the disc. This may suggest the incision in the disc does not produce enough

vascular tissue throughout the disc to influence diffusion of the antibiotic. It also suggests the

capillary network in the endplate is the major source suppling cephazolin to the disc and not

the meagre blood supply in the outer annulus. This was confirmed by the results of cephazolin

concentration in the sheep disc with age (sheep Vs lamb). As the lamb matures the density of

the capillary network diminishes. As this occurs the concentration of cephazolin in the disc

decreases. It appears density of the capillary network is the significant factor influencing the

diffusion of cephazolin into the disc not the vascular supply in the peripheral layers.


Perhaps the uneven distribution of cephazolin in the disc is more pronounced in the lamb disc

due to the increased level of proteoglycans. Or other physiological factors (increased

114

proteoglycan and water content) may provide a nutrient rich environment for bacterial growth

in the nucleus pulposus and may be responsible for an increased infection rate.


Although this was not statistically proven there appears to be a trend that operated lamb discs

were more at risk of discitis than non-operated discs. The only exception was when

cephazolin was given 30 minutes before inoculation. As a consequence of incising the outer

annulus of the disc a vascular granulation tissue response occurs. This response may produce

a favourable environment (increased nutrients) for the growth of bacteria. However, this does

not explain why the same trend is not observed in the sheep. Perhaps the physiological

differences (disc size and vascular supply) or maturity of the immune system between the

sheep and lamb influences the incidence of discitis.


Investigations of discitis using an ovine model have shown that inoculation of the disc with

only a few organisms reliably results in discitis within 1-2 weeks. Previous sheep studies have

looked at the effects of discitis with S. epidermidis none have looked at the effects with S.

aureus. In this study, a large number of bacteria were chosen to ensure the maximal effect of

inoculation was achieved. If the prophylactic antibiotic was efficient in an extreme case then

the results were more credible.


Not all of the organisms were used for inoculation or put into contrast medium. Therefore

bacteria could be counted with or without contrast medium at the time before and after

inoculation.

115


Cellular detail is not shown in Figure 3 as this is a low power view to visualise the extent of

the lesion.


The extraction method was designed and performed by the candidate at the IMVS, Adelaide.

The HPLC assay for cephazolin was only available in Perth and it was necessary that the

samples were assayed there. The candidate spent one week in the Department of Biochemistry

at Royal Perth Hospital and learnt the technique and conducted the antibiotic assay on some

samples.


A large number of bacteria were chosen to ensure the maximal effect of inoculation was

achieved.


Blood cultures at seven days were negative suggesting a large inoculum of bacteria were not

spreading across adjacent discs via the vessels in the highly vascular immature disc or the

vessels surrounding the mature disc.

116

8.5 References 1 Scott JE, Bosworth TR, Cribb AM et al. The chemical morphology of age-related

changes in human intervertebral disc glycosaminoglycans from cervical, thoracic and lumbar nucleus pulposus and annulus fibrosus. JAnat. 1994;184:73-82.

2 Boos N, Weissbach S, Rohrbach H et al. Classification of age-related changes in

lumbar intervertebral discs. Spine 2002; 27:2631-44.

3 Urban JPG, Roberts S, Ralphs JR. The nucleus of the intervertebral disc from development to degeneration. Am Zoo 2000;40:53-61.

4 Urban J. Disc Biochemistry in Relation to Function. second ed. Philadelphia: W.B. Saunders Company, 1996.)

5 Roberts S, Evans H, Trivedi J et al. Histology and Pathology of the Human Intervertebral disc. J Bone Joint Surg (Am) 2006;88:10-14.

6 Rudert M, Tillmann B. Lymph and blood supply of the human intervertebral disc. Cadaver study of correlations to discitis. Acta Orthop Scand 1993;64:37-40.

7 Grunhagen T, Wilde G, Soukane DM et al. Nutrient supply and intervertebral disc metabolism. J Bone Joint Surg (Am) 2006;88:30-35.

8 Maroudas A. Nutrition and Metabolism of the intervertebral disced. Boca Raton, Florida: CRC Press, Inc, 1988

9 Coppes MH, Marani E, Thomeer RT, et al. Innervation of "painful" lumbar discs. Spine 1997;22:2342-9.

10 Wilke H.J, Kettler A, Wenger K.H, et al. Anatomy of the sheep spine and its comparison to the human spine. Anat Rec 1997;247:542-55.

11 Osti OL, Vernon-Roberts B, Fraser RD. 1990 Volvo Award in experimental studies. Anulus tears and intervertebral disc degeneration. An experimental study using an animal model. Spine 1990;15:762-7.

12 Gunzburg R, Fraser RD, Moore R, et al. An experimental study comparing

percutaneous discectomy with chemonucleolysis. Spine 1993;18:218-26. 13 Moore RJ, Osti OL, Vernon-Roberts B, et al. Changes in endplate vascularity after an

outer anulus tear in the sheep. Spine 1992;17:874-8. 14 Fraser RD, Osti OL, Vernon-Roberts B. Iatrogenic discitis: the role of intravenous

antibiotics in prevention and treatment. An experimental study. Spine 1989;14:1025-32.

15 Wilke H.J, Kettler A, Claes L.E. Are Sheep Spines a Valid Biomechanical Model for

Human Spines? Spine 1997;22:2365-74.

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Lumbar Intervertebral Disc Infection: Pathology, Prevention and … · 2007. 9. 5. · Lumbar Intervertebral Disc Infection: Pathology, Prevention and Treatment Rebecca Walters, B