Monday, December 10, 2012

Addressing Pain before it becomes chronic

It is estimated that 50-60% of patients do not receive adequate pain control after surgical or other invasive medical procedures.1,2 Not surprisingly when this pain is not addressed properly there are significant increases in morbidity as well as increases in short-term and long-term medical costs.1,3-9 Some estimate that approximately 116 million individuals suffer from either acute or chronic pain that is not managed properly.10 While there are various, somewhat arbitrary, time periods assigned to the development of chronic pain the general definition is pain that persists beyond the expected period of healing for a given injury. There are two major types of chronic pain: nociceptive, which results due to nocieptor activation (pain receptors) and neuropathic, which results due to damage to the spinal cord or periphery sensory neurons.

One of the initial problems with addressing pain management is the method that is used to evaluate the intensity of pain. While numerous criticisms have been levied against the standard pain numerical reporting system because of its subjective non-uniform nature (a 5 out of 10 for pain for person A may be much different than a 5 out of 10 for person B), little attention is paid to tracking changes in pain progression. Typically post-operative pain is characterized by one or two visits by a nurse with assignment of some pain medication. Basically the pain is viewed as a somewhat static condition that will persist at the recorded level for the duration of the day if not treated by medication. Therefore, one way of better managing pain requires more attentive inquiry regarding how pain is progressing in a patient over the course of a day. Instead of once or twice, inquiries should be made every hour during a normal diurnal time frame for tracking changes. This method will also assist in improving pain management by creating a more reliable evaluation metric for specific treatments. Some seek to measure pain by looking at how certain metabolites change in the bloodstream with time, but with currently limited knowledge of threshold concentrations it is difficult to judge how effective such a strategy would be.

Another problem with pain management is addressing the development of chronic pain. The first question is whether the development of chronic pain stems from improper pain management shortly after surgery or a traumatic event created by improper/inefficient surgery? Based on existing statistics outlining how much long-term pain the population appears to be suffering from it is unlikely that improper/inefficient surgery is the cause of a majority of the chronic pain development. Therefore, this increase in experienced longer-term pain in the population is more than likely due to improper pain management. Unfortunately the current treatment methodology must be flawed in some way because it draws concern that these inconsistencies in effectively dealing with pain arise despite an increase in the overall use of opioids to manage pain.11-14.

While the use of multi-modal analgesia strategies has created sufficient levels of hope in better managing pain, most notably reducing side effects, this hope has not created significantly better long-term outcomes for a majority of people. The inability to attain the potential of multi-modal analgesia is largely due to the large number of variables involved in researching the effectiveness of various technique combinations (dose levels, surgery type, specific genetic factors, analgesic agents, etc.). Basically the individuality demanded by the development and application process reduces a large potential for streamlining and standardizing multi-modal analgesia strategies.

One of the hallmark symptoms of pain is hyperalgesia, which is an increase in pain sensitivity/perception in response to pain inducing stimuli. The typical cause of hyperalgesia is amplification and prolonged nociceptive excitation. Pursuant to this development there is evidence to suggest that current pain management techniques are short-term gain and long-term loss when it comes to addressing pain. One of the most popular choices to managing pain are opioids, especially for acute and chronic cancer pain, but opioids have also started to expand to chronic non-malignant pain. Opioids operate by binding to one of their seventeen different receptors, but three (Mu, Kappa and Delta) are largely responsible for the pain reduction ability of most opioids. Interaction with mu opioid receptors (MOR) either directly or as agonists is the most common pain management pathway. While MORs are expressed on numerous types of neurons the most important with regards to the propagation of pain appear to be the primary sensory neurons called small-sized (C-fiber) and medium sized (A-fiber, specifically Adelta) in the dorsal root ganglia (DRGs).14-17

When molecules interact with MORs they induce presynaptic inhibition that prevents N-type calcium channels from opening, which release neurotransmitters contacting with superficial dorsal horn neurons.18-20 Additional inhibition occurs through activation of G protein-coupled inwardly rectifying potassium channels on dorsal horn neurons resulting in hyperpolarization.20,21 Basically these opioid agonists are effective at addressing pain because of this dual neutralization methodology. Note that there is also some belief of indirect neutralization methods like immune cell activation of opioid receptors.22,23

Unfortunately improper activation of MORs can result in counteracting excitatory activity through the up-regulation of pronociceptive pathways,24,25 which leads to hyperalgesic effects. This specific outcome has been labeled opioid-induced hyperalgesia (OIH). OIH is characterized by increased probabilities for pain in general leading to increased probabilities for the development of chronic pain and tolerance to opioids, which decreases the ability to treat that chronic pain. There is concern that OIH routinely develops into chronic pain due to abrupt inappropriate withdrawals of opioids leading to long-term potentiation (LTP) in the spinal cord. This LTP response is thought to derive from massive activation of NMDA receptor glutamate responses with potential dependency on spinal cord-based TRPV1-expressing afferents along with substance P and chemokines.26-29 OIH can either be acute or chronic.30,31

Focus is applied to spinal cord and DRG LTP because it can develop due to electrical stimulation of appropriate afferents or noxious stimulation (nerve injury or inflammation).27,31-33 One common place for LTP augmentation is at synapses between nociceptive afferents and neurokinin 1 (NK1) receptor expressing projection neurons in lamina I.27 These projection neurons are principally responsible for sending pain signals to the brain.31,34-35 In addition there is similar pharmacology between LTP generation and long-term hyperalgesia.27 Finally LTP development at synapses between C-fibers and superficial dorsal horn neuron is induced by abrupt withdrawal of opioids.26 This is an important distinction because medication in general is typically only administered until symptoms subside. Unfortunately in most situations, including opioid treatment, suddenly stopping medication can result in negative biological consequences.

OIH can ‘leak’ over into the spinal cord by promoting the activation and translocation of protein kinase C, nitric oxide and cholecystokinin and in worst-case scenarios this development can lead to neuronal apoptosis further increasing pain reception problems.36-39 In some respects OIH could be viewed as initially nociceptive and eventually progresses into a neuropathic element.

However, all of this information is still indirect because LTP in the spinal cord with a relation to pain has not been studied directly.27 The lack of direct testing leaves an open question regarding spinal LTP length and how it fully influences the development of chronic pain. LTP for a given group of neurons can last for hours, days, months or a lifetime, but indirect evidence suggests LTP in the spinal cord lasts for several days.27 In this light chronic pain is thought to develop from inhibition of endogenous anti-nociceptive systems or intermittent low-level nociceptive input from periphery neurons. For example pain threshold reduction LTP is also perpetuated to a chronic level through the decreased activity of endogenous anti-nociceptive systems, thus reducing the ‘natural’ abatement adding chronic pain development.

One of tricky elements with addressing OIH is differentiating it from opioid tolerance. When increasing the opioid dosage for treatment of chronic pain the reason for the increase must be identified between opioid tolerance or OIH. In situations of tolerance it may be appropriate to increase opioid concentration depending on the severity of the pain, but in OIH more opioids would result in greater probability of pain. The most common strategy for treating OIH is to cease opioid treatment and substitute a non-opioid analgesic. Unfortunately non-opioid analgesics are typically not as effective as opioids and have their own side effects thus reducing the ability to manage pain.

Differences in analgesic treatment ability has lead to some rotational methodologies where opioids are used for a time and then replaced by non-opioids before a return again to opioids in an attempt to manage pain, but avoid compounding side effects from either treatment. Obviously the success of weaning a patient off of opioids as a means to treat OIH is based on the rate of OIH progression. Unfortunately it is difficult to assess the rate of advancement of OIH in a given patient. However, interestingly enough the future of managing chronic pain may not be developing a new pill or new multi-modal analgesia strategy, but instead developing a strategy where chronic pain does not develop in the first place.

A critical element in the pathway development for OIH is matrix metalloprotease (MMP) concentration. MMPs are a multigene family of tightly regulated zinc-dependent enzymes that maintain homeostasis through their role in tissue degradation and repair.40,41 The two MMPs that appear to play the most prominent roles in pain development are MMP-2 and MMP-9. MMP-9 is frequently released after nerve injury and directs the cleavage of IL-1b.14 Continued cleavage of IL-1b is then governed through a positive feedback mechanism with MMP-2.14,40 There is also suggestion that MMP-9 can interact with NMDA receptors NR1 and NR2B through integrin-beta1 and NO pathways.41 However, MMP-9 influence only seems to occur over a very short time frame (< 24 hrs) for after OIH acquisition to role played by MMP-9 seems to lessen significantly.14

Morphine is one of the most commonly utilized drugs for pain management and is frequently regarded as the standard for comparing the effectiveness of other pain management drugs. Due to its interaction with the μ-opioid receptor morphine chiefly influences in the posterior amygdala, hypothalamus, thalamus, nucleus caudatus and putamen with some associated action in the laminae I and II of the spinal cord. The effects of morphine interaction with its receptor are analgesia and sedation, but can also result in physical dependence.

While morphine is a commonly used pain management drug, its action may have a more detrimental long-term effect in that its interaction with opioid receptors leads to induction of rapid MMP-9 up-regulation. The initial up-regulation occurs in the DRG neurons, not in the spinal cord, and activates pro-nociceptive pathways from the DRG, most notable the cleavage of IL-1b.14 The increased concentration of MMP-9 is not derived from mRNA increases, but translational regulation instead.14 MMP-9 up-regulation does occur in the spinal cord after sustained morphine exposure and could play a role in opioid-induced withdrawal symptoms.41 In some context this biological response could be the body attempting to neutralize the synthetic (non-natural) neutralization of pain possibly in effort to ensure that the mind recognizes that the pain is occurring in effort to cease the pain creating activity, ward off its future application or begin/speed the healing process because pain usually involves some form of injury.

One of the chief aspects of hyperalgesia is the augmentation of Adelta fibers from mechanically insensitive (silent) to mechanically sensitive. This process occurs at high probability in two separate areas: first, during the surgery itself due to cutting an incision and second from MMP-9 up-regulation.42,43 Incision derived hyperalgesia does not rely on NMDA receptor activation, but instead its ‘sister’ receptor a-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA).30,42 This sensitivity increase applies not simply to pain invoking stimuli, but also non-pain inducing mechanical stimuli due to a reduced mechanical response threshold in Adelta fibers.43 Reduced mechanical response also translates into a much larger spontaneous activity (up from 0% to 38% in Adelta afferents and from 0% to 40% in C-fibers).43 This spontaneous activity may play a role in the facilitation of chronic pain through LTP or mechanical sensitization of nociceptors. Inflammation also is though to reduce this spontaneous firing threshold.44,45 In both scenarios the reduced mechanical response threshold decreases gradually to a new equilibrium instead of all at once. This gradual reduction may play a role in the capriciousness of chronic pain development (different people may have different new equilibriums that are obtained at different rates).

Under most circumstances the application of a NMDA antagonist like ketamine can prevent OIH, but such action also reduces the pain neutralization ability of the administered opioid and studies looking at the benefit of combining opioids and NMDA antagonists have resulted in mixed results.46 Include that result with the significant psychotomimetic side effects (sedation, confusion, and lack of coordination) associated with NMDA antagonists and these types of opioid antagonists are typically only used to address opioid overdoses. Part of the problem with using NMDA antagonists to treat pain directly outside of combination with an opioid is that different molecular organizations of the NMDA receptors due to the three different subtypes, each having multiple isoforms, which results in different binding affinities.47

New strategies for short-circuiting the development of the OIH or other chronic pain pathways could be addressed through two different means. First, prevention of IL-1b cleavage, which is a downstream agent in the pain development pathway, will reduce hyperexcitability of sensory neurons by inhibiting potassium channel opening and increasing sodium channel opening.48-50 A similar alternative would be to prevent IL-1b binding by use of a IL-1 receptor antagonist. Second, the elimination of MMP-2 or MMP-9 could treat chronic pain for MMP-2 appears to be a maintenance pain molecule of some sorts whereas MMP-9 seems to be a trigger.

Some believe one strategy to prevent the development of neuropathic pain is to utilize loco-regional anaesthesia techniques over general anaesthesia.30 Some of the loco-regional agents that are hypothesized to be useful are μ-opioid receptor agonists and clonidine along with antagonists at T-type VGCCs and GABAA receptors.27 At least for major morbidities, the data looks promising for the results of several meta-analyses suggest that use of loco-regional analgesia or continuous paravertebral blockade is associated with decreased risk of postoperative pulmonary complications in patients undergoing upper abdominal and thoracic surgical procedures.51,52

The preoperative use of loco-regional analgesia is also associated with a reduction in respiratory complications after major abdominal surgery, although the effect of loco-regional analgesia might not be as prominent as it was previously, partly because the incidence of respiratory complication has progressively decreased during past years.53 Meta-analyses in patients undergoing high-risk cardiothoracic and vascular procedures suggest that use of preoperative thoracic loco-regional analgesia might decrease pulmonary complications, cardiac dysrhythmias, and overall cardiac complications.54,55 So even if current loco-regional analgesia techniques do not have any significant pain reduction characteristics they have some positive benefits.

However, there may be an even better means to amplify loco-regional anaesthesia through the use of MMP-2 and/or 9 inhibitors in the anaesthesia prior to surgery. By preventing MMP-2/9 activity during the pain inducing surgery itself, it may prevent the pain cascade from initiating at any significant level, thus eliminating the need to large amounts of pain control and the potential for the development of OIH. For example NOV manipulation can inhibit MMP-2 expression in the DHSC and MMP-9 expression in DRG and the spinal cord.56 Under normal pain conditions NOV is down-regulated in DRG and DHSC. One means to increase NOV expression is treat individuals with dexamethasone. However, caution must be taken before utilizing the increase of NOV or a similar agent as a treatment possibility because its influence has different effects on different cells. There is little information regarding what negative side effects may stem from applying MMP2/9 inhibitors immediately prior to surgery, so studies must be done to determine their nature and severity. One important consideration is to create the proper balance of inhibition because of the positive role MMP-9 has in wound healing.57

Overall pain management continues to be problematic in society. With the continued increases in OIH development it is more difficult because unless strict controls are established a common means to treat pain can become a catalyst for its further development. Unfortunately patients have a tendency not to be logical and practical when it comes to pain management for when a person is in pain they tend to do stupid things. It could be a great boon to pain management to develop a strategy to neutralize chronic pain before it even fully develops allowing other analgesia elements to be moved to a secondary strategy to treat more extreme conditions. The pre-surgical inhibition of MMP2/9 could have the potential to be such a strategy.


1. Chapman, R, et Al. “Postoperative pain trajectories in cardiac surgery patients.” Pain Research and Treatment. 2012. Article ID 608359. doi:10.1155/2012/608359

2. Wheeler, M, et Al. “Adverse events associated with postoperative opioid analgesia: a systematic review.” Journal of Pain. 2002. 3(3):159–180.

3. Oderda, G, et Al. “Opioid-related adverse drug events in surgical hospitalizations: impact on costs and length of stay.” Ann Pharmacother. 2007. 41:400–06.

4. Ballantyne, J, et Al. “The comparative effects of postoperative analgesic therapies on pulmonary outcome: cumulative meta-analyses of randomized, controlled trials.” Anesthesia and Analgesia. 1998. 86(3): 598–612.

5. Rodgers, A, et Al. “Reduction of postoperative mortality and morbidity with epidural or spinal anaesthesia: results from overview of randomised trials.” The British Medical Journal. 2000. 321(7275):1493–1497.

6. Beattie, W, Badner, N, and Choi, P. “Epidural analgesia reduces postoperative myocardial infarction: a meta-analysis.” Anesthesia and Analgesia. 2001. 93(4):853–858.

7. Holte, K and Kehlet, H. “Effect of postoperative epidural analgesia on surgical outcome.” Minerva Anestesiologica. 2002. 68(4):157–161.

8. Marret, E, Remy, C and Bonnet, F. “Postoperative Pain Forum Group. Meta-analysis of epidural analgesia versus parenteral opioid analgesia after colorectal surgery.” Br J Surg. 2007. 94:665–73.

9. Fischer, H, et Al. “A procedure-specifi c systematic review and consensus recommendations for postoperative analgesia following total knee arthroplasty.” Anaesthesia. 2008. 63:1105–23.

10. Institute of Medicine of the National Academies Report (2011). Relieving Pain in America: A Blueprint for Transforming Prevention, Care Education, and Research. Washington DC: The National Academies Press.

11. Frasco, P, Sprung, J and Trentman, T. “The impact of the joint commission for accreditation of healthcare organizations pain initiative on perioperative opiate consumption and recovery room length of stay.” Anesth Analg. 2005. 100:162–68.

12. Zaslansky, R, et Al. “Tracking the effects of policy changes in prescribing analgesics in one emergency department: a 10-year analysis.” Eur J Emerg Med. 2010. 17:56–58.

13. Manchikanti, L, et Al. “Therapeutic use, abuse, and non-medical use of opioids: a ten-year perspective.” Pain Physician. 2010. 13:401–35.

14. Liu, Y, et Al. “Acute morphine induces matrix metalloproteinase-9 up-regulation in primary sensory neurons to mask opioid-induced analgesia in mice.” Molecular Pain. 2012. 8:19-36.

15. Ji, R, et Al. “Expression of mu-, delta-, and kappa-opioid receptor-like immunoreactivities in rat dorsal root ganglia after carrageenan-induced inflammation.” J. Neurosci. 1995. 15:8156-8166.

16. Wang, H, et Al. “Coexpression of delta- and mu-opioid receptors in nociceptive sensory neurons.” PNAS. 2010. 107:13117-13122.

17. Lee, C, et Al. “Dynamic temporal and spatial regulation of mu opioid receptor expression in primary afferent neurons following spinal nerve injury.” Eur J. Pain. 2011. 15:669-675.

18. Heinke, B, Gingl, E, and Sandkühler, J. “Multiple Targets of mu-Opioid Receptor-Mediated Presynaptic Inhibition at Primary Afferent A{delta}- and C-Fibers.” J. Neurosci. 2011. 31:1313-1322.

19. Kohno, T, et Al. “Actions of opioids on excitatory and inhibitory transmission in substantia gelatinosa of adult rat spinal cord.” J. Physiol. 1999. 518(3):803-813.

20. Kohno, T, et Al. “Peripheral axonal injury results in reduced mu opioid receptor pre- and post-synaptic action in the spinal cord.” Pain. 2005. 117:77-87.

21. Yoshimura, M, North, R. “Substantia gelatinosa neurones hyperpolarized in vitro by enkephalin.” Nature. 1983. 305:529-530.

22. Mousa, S, et Al. “Beta-Endorphin-containing memory-cells and mu-opioid receptors undergo transport to peripheral inflamed tissue.” J. Neuroimmunol. 2001. 115:71-78.

23. Stein, C, et Al. “Peripheral mechanisms of pain and analgesia.” Brain Res Rev. 2009. 60:90-113.

24. Angst, M, Clark, J. “Opioid-induced hyperalgesia: a qualitative systematic review.” Anesthesiology. 2006. 104:570-587.

25. Mao, J, Price, D, and Mayer, D. “Mechanisms of hyperalgesia and morphine tolerance: a current view of their possible interactions.” Pain. 1995. 62:259-274.

26. Drdla, R, et Al. “Induction of synaptic long-term potentiation after opioid withdrawal.” Science. 2009. 325:207-210.

27. Ruscheweyh, R et Al. “Long-term potentiation in spinal nociceptive pathways as a novel target for pain therapy.” Molecular Pain. 2011. 7:20-57.

28. Chen, Y, Geis, C, and Sommer, C. “Activation of TRPV1 contributes to morphine tolerance: involvement of the mitogen-activated protein kinase signaling pathway.” J. Neurosci. 2008. 28:5836-5845.

29. Ma, W, et Al. “Morphine treatment induced calcitonin gene-related peptide and substance P increases in cultured dorsal root ganglion neurons.” Neuroscience. 2000. 99:529-539.

30. Wu, C and Raja, S. “Treatment of acute postoperative pain.” The Lancet. 2011. 377:2215–25.

31. Ikeda, H, et Al. “Synaptic amplifier of inflammatory pain in the spinal dorsal horn.” Science. 2006. 312:1659-1662.

32. Zhang, H, et Al. “Acute nerve injury induces long-term potentiation of C-fiber evoked field potentials in spinal dorsal horn of intact rat.” Sheng Li Xue Bao. 2004. 56:591-596.

33. Sandkühler, J and Liu, X. “Induction of long-term potentiation at spinal synapses by noxious stimulation or nerve injury.” Eur J Neurosci. 1998. 10:2476-2480.

34. Nichols, M, et Al. “Transmission of chronic nociception by spinal neurons expressing the substance P receptor.” Science. 1999. 286:1558-1561.

35. Mantyh, P, et Al. “Inhibition of hyperalgesia by ablation of lamina I spinal neurons expressing the substance P receptor.” Science. 1997. 278:275-279.

36. Mayer, D, et Al. “Cellular mechanisms of neuropathic pain, morphine tolerance, and their interactions.” PNAS. 1999. 96:7731– 6.

37. Chen, L and Huang, L. “Sustained potentiation of NMDA receptormediated
glutamate responses through activation of protein kinase C by u-opioids.” Neuron. 1991. 7:319 –26.

38. Chen, L, and Huang, L. “Protein kinase C reduces Mg2+ block of NMDA-receptor channels as a mechanism of modulation.” Nature. 1992. 356:521–3.

39. Mao, J, Price, D and Mayer, D. “Thermal hyperalgesia in association with the development of morphine tolerance in rats: roles of excitatory amino acid receptors and protein kinase C.” J. Neurosci. 1994. 14:2301–12.

40. Ribeiro, A, et Al. “Expression of matrix metalloproteinases, type IV collagen, and interleukin-10 in rabbits treated with morphine after lamellar keratectomy.” Veterinary Ophthalmology. 2012. 15(3):153-163.

41. Liu, W, et Al. “Spinal matrix metalloproteinase-9 contributes to physical dependence on morphine in mice.” J. Neurosci. 2010. 30:7613-7623.

42. Zahn, P, Umali, E and Brennan, T. “Intrathecal non-NMDA excitatory amino acid receptor antagonists inhibit pain behaviors in a rat model of postoperative pain.” Pain. 1998. 74:213–23.

43. Pogatzki, E, Gabhart, G and Brennan, T. “Characterization of Adelta- and C-Fibers Innervating the Plantar Rat Hindpaw One Day After an Incision.” J. Neurophysiol. 2002. 87:721-731.

44. Ahlgren, S, White, D and Levine, J. “Increased responsiveness of sensory neurons in the saphenous nerve of the streptozotocin-diabetic rat.” J Neurophysiol. 1992. 68:2077–2085.

45. Kocher, L, et Al. “The effect of carrageenan-induced inflammation on the sensitivity of unmyelinated skin nociceptors in the rat.” Pain. 1987. 29:363–373.

46. Van Elstraete, A, et Al. “A Single Dose of Intrathecal Morphine in Rats Induces Long-Lasting Hyperalgesia: The Protective Effect of Prior Administration of Ketamine.” Anesth Analg. 2005. 101:1750 –6.

47. Paoletti, P and Neyton, J. “NMDA receptor subunits: function and pharmacology.” Curr Opin Pharmacol. 2007. 7(1):39–47.

48. Takeda, M, et Al. “Enhanced excitability of nociceptive trigeminal ganglion neurons by satellite glial cytokine following peripheral inflammation.” Pain. 2007. 129:155-166.

49. Binshtok, A, et Al. “Nociceptors are interleukin-1beta sensors.” J. Neurosci. 2008.

50. Takeda, M, et Al. “Activation of interleukin-1beta receptor suppresses the voltage-gated potassium currents in the small-diameter trigeminal ganglion neurons following peripheral inflammation.” Pain. 2008. 139:594-602.

51. Marret, E, et Al. “Meta-analysis of intravenous lidocaine and postoperative recovery after abdominal surgery.” Br J Surg. 2008. 95:1331–38.

52. Hudcova, J, et Al. “Patient controlled opioid analgesia versus conventional opioid analgesia for postoperative pain.” Cochrane Database Syst Rev. 2006. 4:CD003348.

53. Wijeysundera, D, et Al. “Epidural anaesthesia and survival after intermediate-to-high risk non-cardiac surgery: a population-based cohort study.” Lancet. 2008. 372:562–69.

54. Wu, C, et Al. “Effect of postoperative epidural analgesia on morbidity and mortality following surgery in medicare patients.” Reg Anesth Pain Med. 2004. 29:525–33.

55. Liu, S and Wu, C. “Effect of postoperative analgesia on major postoperative complications: a systematic update of the evidence.” Anesth Analg. 2007. 104:689–702.

56. Kular, L, et Al. “NOV/CCN3 attenuates inflammatory pain through regulation of matrix metalloproteinases-2 and –9.” Journal of Neuroinflammation. 2012. 9:36-55.

57. Broadbent, E, et Al. “Psychological stress impairs early wound repair following surgery.” Psychosomatic Medicine. 2003. 65:865-869.

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