lumbar intervertebral disc infection: pathology, prevention and … · 2007. 9. 5. · lumbar...
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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.
7
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
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17
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18
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19
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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
3.1 Statement of authorship
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
3.2 Title page for published manuscript
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
52
Chapter 4: Prophylactic cephazolin to prevent discitis in an ovine model
4.1 Statement of authorship
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
4.2 Title page for published manuscript
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
3The Spinal Unit, Royal Adelaide Hospital, Adelaide, Australia
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
NOTE: This publication is included on pages 53 – 68 in the print copy of the thesis held in the University of Adelaide Library.
69
Chapter 5: Preventing and treating discitis: Cephazolin penetration in ovine lumbar intervertebral disc
5.1 Statement of authorship
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
5.2 Title page for published manuscript
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
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
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
85
Chapter 6: Penetration of cephazolin in human lumbar intervertebral disc
6.1 Statement of authorship
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
6.2 Title page for published manuscript
Chapter 6
PENETRATION OF CEPHAZOLIN IN HUMAN LUMBAR INTERVERTEBRAL DISC
R.M. Walters1, 2, 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 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
NOTE: This publication is included on pages 86 – 99 in the print copy of the thesis held in the University of Adelaide Library.
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.
8.4.2 Chapter 2 page 27, paragraph 1
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.
8.4.3 Chapter 2 page 28, paragraph 1
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.
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8.4.4 Chapter 2 page 28, paragraph 1
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.
8.4.5 Chapter 2 page 28, paragraph 2
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).
8.4.6 Chapter 2 page 29, paragraph 2
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).
8.4.7 Chapter 2 page 29, paragraph 2
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.
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8.4.8 Chapter 3 page 44, paragraph 3
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.
8.4.9 Chapter 3 page 46, paragraph 4
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.
8.4.10 Chapter 3 page 46, paragraph 4
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.
8.4.11 Chapter 3 page 46, paragraph 3
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
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the disc is restricted by the repulsion of the like charges. Therefore concentration of
cephazolin in the outer disc (annulus pulposus) remains greater.
8.4.12 Chapter 4 page 59, paragraph 2
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.
8.4.13 Chapter 4 page 64, paragraph 3
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.
8.4.14 Chapter 4 page 64, paragraph 1
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
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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.
8.4.15 Chapter 4 page 64, paragraph 3
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.
8.4.16 Chapter 4 page 63, paragraph 1
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.
8.4.17 Chapter 4 page 59, paragraph 1
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.
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8.4.18 Chapter 4 page 60, paragraph 3
Cellular detail is not shown in Figure 3 as this is a low power view to visualise the extent of
the lesion.
8.4.19 Chapter 4 page 65, paragraph 1
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.
8.4.20 Chapter 4 page 61, paragraph 2
A large number of bacteria were chosen to ensure the maximal effect of inoculation was
achieved.
8.4.21 Chapter 5 page 79, paragraph 3
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.
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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.
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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.
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8 Maroudas A. Nutrition and Metabolism of the intervertebral disced. Boca Raton, Florida: CRC Press, Inc, 1988
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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.