ISCEV Standard for Clinical Electro-oculography -LRB- EOG -RRB- 2006 The Clinical Electro-oculogram -LRB- EOG -RRB- is an electrophysiological test of function of the outer retina and retinal pigment epithelium -LRB- RPE -RRB- in which the change in the electrical potential between the cornea and the ocular fundus is recorded during successive periods of dark and light adaptation . This document sets out a Standard Method for performance of the test , and also gives detailed guidance on technical and practical issues , and on reporting test results . The main object of the Standard is to promote consistent quality of testing and reporting within and between centres . This 2006 Standard , from the International Society for Clinical Electrophysiology of Vision -LRB- ISCEV : www.iscev.org -RRB- , is a revision of the previous Standard published in 1993 , and reviewed and re-issued in 1998 . Purpose and use of this Standard This Standard is one of a series of Standards and Guidelines for electrophysiology of vision -LSB- 1 -- 8 -RSB- available for download from www.iscev.org , and is a revision of the ISCEV Standard for Clinical Electro-oculography -LRB- first issued 1993 -- re-approved 1998 -RRB- -LSB- 1 , 2 -RSB- , which provides a Standard Method for measurement of the electro-oculogram -LRB- EOG -RRB- . Also included is advice on possible variations on the Standard Method , additional tests , and comment on the procedures recommended . The major change in this current standard compared with the earlier EOG standards is that it recommends one single standard testing protocol and measurement strategy . Clinical and research users of the clinical EOG are encouraged to use the current Standard Method where possible , to achieve consistency of results within and between test centres . Reports of EOG recordings performed to the Standard Method given here should cite this 2006 Standard . Where a method is used which deviates from the Standard Method , the deviations should be stated , together with any normative or reference data . Where the method used conforms to a previous EOG Standard , this may be cited instead . EOG origins , pathological effects , and principles of measurement Electrophysiology of the RPE in dark and light adaptation The eye has a standing electrical potential between front and back , sometimes called the corneo-fundal potential . The potential is mainly derived from the retinal pigment epithelium -LRB- RPE -RRB- , and it changes in response to retinal illumination . The potential decreases for 8 -- 10 min in darkness . Subsequent retinal illumination causes an initial fall in the standing potential over 60 -- 75 s -LRB- the fast oscillation -LRB- FO -RRB- -RRB- , followed by a slow rise for 7 -- 14 min -LRB- the light response -RRB- . These phenomena arise from ion permeability changes across the basal RPE membrane . The clinical electro-oculogram -LRB- EOG -RRB- makes an indirect measurement of the minimum amplitude of the standing potential in the dark and then again at its peak after the light rise . This is usually expressed as a ratio of ` light peak to dark trough ' and referred to as the Arden ratio . The behaviour of the corneo-fundal potential in the normal eye is predictable in defined conditions , such as those described in this Standard , but changing from dark to light actually initiates a triggered response extending for about 2 h in the form of a diminishing sinusoidal oscillation . Diseases affecting the light response of the EOG The light response is affected in diffuse disorders of the RPE and the photoreceptor layer of the retina including some characterised by rod dysfunction , or chorio-retinal atrophic and inflammatory diseases . In most of these there is correlation with the electroretinogram -LRB- ERG -RRB- , except notably in the case of Best 's vitelliform maculopathy , in which the clinical EOG is usually highly abnormal in the presence of a normal ERG . Measurement of the clinical EOG The potential across the RPE causes the front of the eye to be electrically positive compared to the back . As a result , potentials measured between two electrodes placed on the skin at each side of an eye will change as the eye turns from left to right . The EOG method is used widely to record eye movements , on the assumption of unchanging corneo-fundal potentials . In the clinical EOG described here , we use defined eye movements to monitor the changes in corneo-fundal potential . If the test subject looks alternately at targets a fixed angle apart , the potential recorded from the skin resembles a square wave whose amplitude will be a fixed proportion of the corneo-fundal potential . During a light/dark cycle , this indirectly measured potential will change in the same way as the source potentials , so that the Arden ratios -LRB- and timing of peaks etc. -RRB- will be a close approximation to the average changes occurring across the RPE . The Standard Method This section outlines the Standard Clinical EOG method , definitions , explanations , and instrument specifications . Further notes regarding testing strategies are given in later sections . Pupils Apply dilating drops before any other action to allow pupil dilation to be relatively complete by the start of the test -LRB- see notes below -RRB- . Their size should be checked at the start and end of the test and recorded at the end of the test . Electrodes After suitable skin preparation , place small recording electrodes , close to the canthi of each eye as in Fig. 1 . Connect the electrodes from each eye to separate channels of a differential amplifier . The ` ground ' electrode can be placed on the forehead . The impedance between any pair of electrodes should not exceed 5 kΩ . The electrodes , amplifier and impedance meter must be approved for medical use . Fig. 1 -LRB- a and b -RRB- . Recording electrode positions Amplifier This should have a band pass of either 0 -LRB- d.c. -RRB- to 30 Hz , or 0.1 to 30 Hz , to provide recordings of the saccades which appear as square waves . Full field -LRB- Ganzfeld -RRB- stimulator This should have a comfortable head/chin rest , and two red fixation lights 15 degrees left and right of centre . The fixation lights should be bright when the light adapting background is on , and as dim as practical in the dark . Pre-adaptation The test subject should be in stable indoor lighting for as long as possible before the test , and should not be exposed to any large changes in lighting -LRB- lighter/darker -RRB- during this period , such as indirect ophthalmoscopy . As near as is practical , the pre-test light exposure should be the same for all test subjects . Preparing the test subject Explain the procedure including , ` chin/head on rest/restraint in stimulator , 15 min dark , 15 min light , fixation lights alternate in simple rhythmic manner , for 10 s each minute , when lights change , move eyes in single sweep to next one , do not turn head , do not anticipate the changes ' . Practice the procedure with the recording system on and coach the subject if there is head movement , overshoot , stepping , or anticipation . Dark phase In total darkness for 15 min , except for the dim fixation lights , alternate the fixation lights every 1 s for 10 s every 1 min , and record the resulting EOG potentials . The test subject should remain looking into the stimulator the whole time if possible , and should be warned of the start of each measurement sequence to ensure attention . The operator must have an instant view of the recordings to check for patient compliance , and errors such as overshoot . Light phase Bring on the ganzfeld background light of 100 photopic cd/m2 . If necessary , bring this on gradually over a short period -LRB- e.g. 20 s -RRB- for patient comfort , especially in cases of photophobia . Continue recording every 1 min as above . Keep the test subject forward in the stimulator bowl for the whole time and with eyes open . This completes the procedure for the test subject . Measure the amplitude of the EOG Taking care to remove the effects of overshoot -LRB- see Fig. 2 and notes below -RRB- and stepped saccades , measure the EOG amplitude in μV , either manually or by a computer algorithm . Calculate the average of the amplitudes in each 10 s trial . Fig. 2Idealised saccadic recording with d.c. amplifier -LRB- top -RRB- and example a.c. coupled amplifier with high pass filter at 0.5 Hz and 0.1 Hz . Overshoot is hard to recognise using 0.5 Hz Plot the change in the responses Plot the average amplitude of the EOG in μV on a graph representing the 30 min of the test . Ignore any points known to be incorrect -LRB- e.g. subject did not follow the fixation lights properly -RRB- , and identify the underlying curve -LRB- see notes -RRB- as in Fig. 3 -LRB- lower panel -RRB- . Fig. 3Idealised -LRB- underlying -RRB- EOG response -LRB- top -RRB- and practical response with noise and trial/trial variability . Arrows show the dark trough -LRB- DT -RRB- and light peak -LRB- LP -RRB- . The underlying curve must be estimated before recording the Arden ratio -LRB- LP/DT -RRB- Calculate the Arden ratio This is the ratio between the peak and trough of the underlying curve , not the maximum and minimum recorded values . Report As a basic factual report state the Arden ratio , the first -LRB- dark -RRB- trough amplitude in μV , the time from start of the light phase to the peak -LRB- if present -RRB- , the pupil size , and the type of adapting light source . The report should also state any difficulties which were encountered during the test session which may affect confidence in the results , such as the test subject 's difficulty in performing the test . Deviation from the Standard This Standard represents a basic or core procedure . If a laboratory chooses a procedure which varies from the Standard Method above , it is critical to cite this Standard , but specify the deviations from the Standard Method , such as different luminance level for the adapting light . If a statistical report is given , then this must be supported by reference data obtained under the same conditions . Additional tests Some centres measure the ` Fast Oscillation ' -LRB- FO -RRB- , often in conjunction with the Clinical EOG . The fast oscillations : The fast oscillations -LRB- FOs -RRB- have the opposite polarity to the light rise of the Standard Clinical EOG . At light onset there is a decrease in the standing potential that recovers 30 -- 40 s after light onset . This decrease , the ` light-trough ' -LRB- LT -RRB- , is caused by a decrease in intracellular Cl - that results in a decrease of Cl - transport across the basolateral membrane resulting in a hyperpolarisation of the RPE basal membrane . The FO is recorded using the same parameters as for the clinical EOG -LRB- amplification , electrode placement and stimulus light intensity -RRB- . However , recordings should be made continuously as the subject executes regular horizontal saccades at 1/s . Alternating light and dark for 60 s each induces the FOs , which have a near sinusoidal appearance . During the light interval a light trough -LRB- LT -RRB- develops and begins to rise again after 30 -- 40 s . The subsequent interval of darkness results in a dark rise -LRB- DR -RRB- at 30 -- 40 s following the onset of darkness . The next interval of light induces another light trough . The total number of light-dark intervals should be at least 4 with 60 s periods of light and dark , making a total test time of 8 min . Pre-adaptation does not affect the FO , and so this test can be performed either independently or in conjunction with the clinical EOG , provided pre-adaptation conditions for the latter are consistent within the lab . Figure 4 shows a schematic idealised representation of the FO cycle , from which the ratio of DP : LT should be reported . The normal amplitude ratio is typically between 1.05 and 1.30 . However , at present each laboratory should obtain and use their own reference data . It should be noted that high blood glucose levels increase the FO amplitudes . Fig. 4Idealised representation of fast oscillations -LRB- FO -RRB- . In the dark intervals -LRB- black bars -RRB- the standing potential increases to a dark rise maximum -LRB- DR -RRB- . Following light onset the standing potential falls to a light trough -LRB- LT -RRB- . The FO ratio of the DR : LT standing potentials should be recorded Practical notes , instruments and definitions -LRB- alphabetical -RRB- Amplifiers For a 30 degree saccade , the typical EOG amplitudes are between 250 and 1000 μV with essential frequency content of 0 to 30 Hz . With such signal amplitudes , no special shielding nor interference protection should be necessary . Medical grade amplifiers intended for human physiological recording are required to be ` electrically isolated ' to prevent any risk of electric shock to the patient via the recording electrodes . Laboratory grade amplifiers may not have this protection . To obtain recordings of saccadic movements as a ` square wave ' -LRB- the ideal -RRB- requires a d.c. amplifier -LRB- i.e. no high pass filter -RRB- but there are operational penalties in terms of base line shift due to electrode polarisation and movement , which can be partially eliminated by the use a high pass filter -LRB- e.g. 0.1 Hz -RRB- . If a higher frequency is used -LRB- e.g. 0.5 Hz -RRB- there is considerable ` droop ' of the square wave , making identification of overshoot and stepped saccades more difficult -LRB- see Fig. 2 -RRB- . For the low pass filter , 30 Hz is sufficient . Including higher frequencies adds noise to the response -LRB- e.g. EMG -RRB- and since most automatic cursor placement regimes find the lowest and highest parts of the wave , this exaggerates the recorded amplitudes . Since both the peak and the trough amplitudes will be equally exaggerated the Arden ratio will be underestimated . For guidance on verifying amplifier performance , see reference -LSB- 3 -RSB- . Amplifier saturation EOG potentials measured during saccadic eye movements as in the Standard Clinical Method can vary by about 5:1 in amplitude between subjects , which , with the light rise , may mean a total amplitude range of up to 15:1 . Thus , the operator must be able to see the recordings of the saccades to ensure saturation does not occur , and to adjust the amplifier gain settings accordingly . Arden ratio Using the Standard clinical EOG method described here , the Arden ratio is the peak EOG amplitude occurring in the light phase , divided by the minimum amplitude during the dark phase . The peak and trough values must be measured from the ` underlying curve ' as shown on Fig. 3 , not from individual measurements . Compliance of the patient Test subjects will have difficulty performing the saccadic movements if they do not have good central vision , or suffer from diplopia or dysconjugate eye movements , have nystagmus , are very young , or are infirm and are unable to take up the physical positions required for the duration of the tests . Some patients also suffer claustrophobia or fear of the dark , and so the testing must be performed in such a way as to minimise these fears . Real-time view of the EOG potentials , and infrared camera view of the patient 's eyes , are very useful in ensuring full compliance . In most cases , coaching under observation can remedy poor co-operation . A common issue is the patient closing eyes during the light phase , which can be seen by a camera . The cost of unreliable performance is that the margins of error are widened . This does not mean that no information can be gleaned . If the suspected disorder is one with a clear test outcome , it may be possible to make the diagnosis on one or two reliable trials performed near the time of the trough and the peak , to ensure that there is indeed a light rise of some amplitude , or not . Diplopia Test subjects often find the fixation lights too near for them to achieve convergence . The subject may be advised to look between the pair of images in cases of intractable diplopia . Subjects with ocular motility problems are likely to give unreliable results . If the suspected retinal disorder is bilateral , it is acceptable to patch one eye to perform the tests -LRB- see the notes on interaction between eyes below -RRB- . Electrodes Recording the EOG is relatively undemanding as regards the electrodes . These should be relatively non-polarisable such as standard medical EEG or ECG electrodes , of a size appropriate for attachment to the side of the nose . If baseline drift is excessive , less polarisable electrodes may be necessary . Fixation targets These should be small , red , and adjustable in brightness so that they can be seen clearly by all subjects above the background illumination , and to be dim -LRB- just visible to the test subject -RRB- in darkness . Full field -LRB- Ganzfeld -RRB- stimulator This should be as large as practicable to allow adequate distance from eye to fixation lights . It should have a chin rest and forehead bar to ensure stable head position , and it should have fixation lights 15 degree each side of centre . The background intensity should be stable and ` visibly white ' -LRB- see notes on Light below -RRB- . Interaction between eyes The EOG potentials from one eye will contaminate the response from the other . This is approximately 15 % with electrodes placed on each side of the nose close to the inner canthi , rising to about 40 % as they come close together and then touch -LRB- e.g. becoming a common central electrode placed on the bridge of the nose -RRB- . This can give misleading results in cases of an electrically inactive -LRB- e.g. total retinal detachment -RRB- , or absent eye , in which case the defective eye appears to have the same Arden ratio as for the fellow eye , albeit on a much smaller apparent standing potential . More confusing is when the eyes have similar standing potentials but different Arden ratios . In these cases , the measured Arden ratio from the better eye is enhanced at the expense of that from the weaker eye , which can in fact appear to have an Arden ratio of less than 1.0 , solely due to interaction . Light Luminance For the light phase the luminance of the full field adapting light should be 100 photopic cd/m2 as measured with a photometer -LRB- photopic filter -RRB- with a traceable calibration -LRB- see also notes on pupil dilation below -RRB- . The calibration of the ganzfeld stimulator should be carried out periodically , e.g. once a year , and corrective action applied -LRB- see ref -LSB- 3 -RSB- for guidance -RRB- . Modest room lighting may be turned on during the light phase when the impact on the effective luminance in the bowl should be negligible with the test subject looking into the bowl . Darkness The dark phase should take place in total darkness , and the fixation lights should be dimmed to the minimum necessary to enable fixation . Colour There are several possible sources of adapting light such as tungsten , halogen , LED , and fluorescent . For a commercial recording system , the type will be stated in the manufacturer 's literature . Spectrally these are quite different , and the spectra for tungsten and halogen also change with brightness . This issue makes the definition of a single Standard Method for recording the clinical EOG difficult at present , and therefore all reports should state the type of light source used . If the user has additional data , such as colour temperature , or scotopic luminance , then this should be stated . Normative data/reference range At present there is no standard international reference range for the clinical EOG . With the introduction of this Standard -LRB- The Standard Clinical EOG Method 2006 -RRB- there is a greater possibility of developing and comparing normative data -LRB- see notes on reporting -RRB- . Photophobia The EOG ` light rise ' is a ` triggered ' response , and is assumed to start from the onset of the light phase . Some test subjects will find the light too bright and it may be necessary to bring the adapting light up gradually over , say , a 20-second period . If so , this variation should be stated . If the adapting light is brought on very slowly -LRB- e.g. minutes -RRB- , the response is changed . Plotting The average EOG amplitude calculated from each 10-second trial should be plotted -LRB- see Fig. 3 -RRB- . The first critical step is that the underlying physiologic curve is recognised and drawn for derivation of the trough and peak . This may require a curve fitting algorithm or a traditional ` flexicurve ' -LRB- curve fitting ruler -RRB- on paper . Only then should the Arden ratio be calculated . It is helpful if any uncertain values have been identified and marked at the time of recording , so that they can be ignored when identifying the underlying curve . Pupil dilation Having dilated pupils means less variability in the light entering the eye . There may be occasions when full pupil dilation is impossible or undesirable . If pupils are not artificially dilated , then the report should state this . An attempt should be made to adjust the adapting luminance to accommodate smaller or variable pupils , and this luminance should be quoted -LRB- it is usual to try to achieve the same value in Trolands , as for dilated pupils -RRB- . In many cases useful diagnostic information can be obtained without dilated pupils , especially with extreme results -LRB- no light rise , strong light rise -RRB- . Intermediate results for the Arden ratio will obviously be of less value with undilated and therefore variable pupil sizes . Reporting Basic factual report This should include the Arden ratio , the first -LRB- dark -RRB- trough amplitude in μV , the time from the start of the light phase to the light peak -LRB- if present -RRB- , the pupil size at the end of the test , and the type of adapting light source . Statistical report This should show how the calculated Arden ratio relates to a quoted reference range . If the reference range was obtained with any different test parameters , this should be explained . No fully authenticated normal reference data is available currently for the Standard Clinical EOG method given above . However , from a review of existing published data , Arden ratios < 1.5 are reported as being abnormally low , and those > 2.0 are reported as probably normal , and in between as borderline . This guidance may be useful in the absence of local normative data , but the values do not constitute validated diagnostic criteria . Saccade measurement Use a scale or electronic calliper to measure the change in EOG potential resulting from each saccade , and calculate an average for each 10 second trial . The average should include only those measurements judged to be reliable . Alternative measurement methods may be used , including computer algorithms . If a computer algorithm is used there is a need to ensure that the values returned properly represent the true EOG amplitudes . Each algorithm is likely to make some mistakes . Fortunately , the final dark/light response curve will form an ` average of averages ' in which the influence of a few errors should be swamped by the remaining data . Particular causes of unreliability are overshoot -LRB- see Fig. 2 -RRB- , stepped saccades , missing saccades , inverse saccades -LRB- eyes go opposite way to fixation lights -RRB- , and eccentric fixation in which the saccade length switches between two or more values . When measuring manually , these difficulties are fairly obvious . Standing potentials This Standard recommends the reporting of the minimum standing potential -LRB- dark trough minimum -RRB- , taken from the underlying response curve , not the minimum recorded value . This value is not often used in diagnosis at present , but if the value is abnormally low it may indicate an inactive retina -LRB- e.g. total retinal detachment -RRB- , and the calculated Arden ratio may be unreliable because of the low value of the divisor in the ratio . Normal values for the minimum standing potential should be established , but as a guide , minimum standing potentials less than 150 μV should be treated as low , and more likely to produce unreliable Arden ratios . Warning of start of each trial There should be a warning , verbal or automatic , of the impending start of each trial , to ensure readiness of both test subject and operator . Some users have sounds -LRB- beeps -RRB- to co-incide with the alternations of the fixation lights . However , it may be better to rely solely on the visual stimuli , to avoid the possibility of alternation without actual fixation . History and acknowledgements This Standard forms part of series of Standards , Recommendations and Guidelines prepared by the International Society for Clinical Electrophysiology of Vision -LRB- ISCEV -RRB- : Participants in the review : ISCEV EOG Standard Revision Committee 2006 ; Malcolm Brown -LRB- Chairman -RRB- , Royal Liverpool University Hospital , Liverpool UK Michael Marmor , Stanford University , California USA ; Vaegan -LRB- ISCEV Board Member-at-Large -RRB- University of New South Wales , Australia , Eberhart Zrenner University Eye Hospital , Tuebingen , Germany EOG Standard Advisory Panel 2006 includes the members above plus : Michael Bach , -LRB- ISCEV President -RRB- , Universität-Augenklinik , Freiburg , Germany : Mitchell Brigell , -LRB- ISCEV Director of Standard -RRB- , Pfizer Global . R&D , Ann Arbor , Michigan USA : Graham Holder , -LRB- ISCEV Director of Education -RRB- , Moorfields Eye Hospital , London UK Paul Constable , City University , London UK : Masao Yoshikawa , Mayo Corporation , Aichi , Japan ; Carol Westall , Hospital for Sick Children , Toronto Canada ; Geoffrey Arden , -LRB- Honorary ISCEV member -RRB- City University , London UK

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

arden ratio
light adaptation
retinal pigment epithelium


Increased osteoclastic activity in acute Charcot 's osteoarthopathy : the role of receptor activator of nuclear factor-kappaB ligand Aims/hypothesis Our aims were to compare osteoclastic activity between patients with acute Charcot 's osteoarthropathy and diabetic and healthy controls , and to determine the effect of the receptor activator of nuclear factor-kappaB ligand -LRB- RANKL -RRB- and its decoy receptor osteoprotegerin -LRB- OPG -RRB- . Introduction Although Charcot 's osteoarthropathy is characterised by increased local bone resorption -LSB- 1 -RSB- , the exact cellular mechanisms contributing to the pathogenesis of this condition remain unresolved . Osteoclasts have been shown to be the principal cell type responsible for bone resorption -LSB- 2 -RSB- . These cells originate from the haemopoietic lineage and are known to undergo various stages of proliferation , fusion and differentiation before they are fully functionally active , mature osteoclasts . Recently , receptor activator of nuclear factor-kappaB -LRB- RANK -RRB- ligand -LRB- RANKL -RRB- has been identified as an essential mediator of osteoclast formation and activation -LSB- 3 -RSB- . RANKL is expressed on a variety of cell types such as bone forming osteoblasts , T lymphocytes , dendritic cells , endothelial cells and fibroblasts . RANKL mediates the process of osteoclastogenesis by binding to its RANK , which is expressed on mononuclear osteoclast precursors . The effects of RANKL -- RANK interaction are physiologically counterbalanced by osteoprotegerin -LRB- OPG -RRB- , which acts as a soluble receptor decoy for RANKL and blocks the interaction of RANKL with RANK . The ratio of RANKL to OPG has been suggested to regulate the extent of osteoclast formation and resorption . Therefore , any alteration in the RANKL/OPG ratio could be critical in the pathogenesis of osteolytic bone disorders -LSB- 4 -RSB- . Recently , Jeffcoate hypothesised that the RANK/RANKL/OPG pathway may play an important role in the osteolysis seen in acute Charcot 's osteoarthropathy -LSB- 5 -RSB- . Using an in vitro technique to generate functional human osteoclasts from peripheral blood monocytes -LRB- PBMCs -RRB- -LSB- 6 -RSB- in the presence of macrophage-colony stimulating factor -LRB- M-CSF -RRB- -LSB- 7 -RSB- and soluble RANKL , it is possible to determine the cellular mechanisms involved in the process of osteoclast formation and resorption in physiological and pathological conditions . To our knowledge , this technique has not yet been studied in patients with Charcot 's osteoarthropathy . The aims of this study were : -LRB- 1 -RRB- to generate functional human osteoclasts in vitro from diabetic patients with acute Charcot 's osteoarthropathy and from healthy and diabetic control participants ; -LRB- 2 -RRB- to compare the extent of osteoclast formation and resorption ; and -LRB- 3 -RRB- to determine the role of the RANK/RANKL/OPG pathway in osteoclastic activity in Charcot 's osteoarthropathy . Methods Patients We studied nine consecutive diabetic patients with recent onset of acute Charcot 's osteoarthropathy -LRB- five men , four women ; five type 1 , four type 2 diabetes -RRB- , eight diabetic patients with no previous history of Charcot 's osteoarthropathy -LRB- five men , three women ; four type 1 , four type 2 -RRB- and eight healthy control participants -LRB- five men , three women -RRB- . Patients with acute Charcot 's osteoarthropathy were matched for age and duration of diabetes with the diabetic control patients and for age with the healthy control participants . The mean age was similar between patients with Charcot 's osteoarthropathy and diabetic control patients -LRB- 53 ± 2.8 versus 59 ± 2.9 years -LSB- mean ± SEM -RSB- , p = 0.167 -RRB- as was the mean age between the former and healthy control participants -LRB- 53 ± 2.8 versus 47 ± 2.7 years , p = 0.114 -RRB- . The mean duration of diabetes was similar in both groups with diabetes -LRB- 31 ± 5.1 -LSB- Charcot patients -RSB- versus 27 ± 4.6 years , p = 0.606 -RRB- . Diabetes control as indicated by glycated Hb was also similar in the two diabetes groups -LRB- 7.7 ± 0.6 -LSB- Charcot 's -RSB- versus 7.8 ± 0.4 % , p = 0.743 -RRB- . Diagnosis of Charcot 's osteoarthropathy was made on the presentation of a hot swollen foot , with skin foot temperature 2 °C greater than the corresponding site on the contralateral foot and with typical radiological changes of subluxation , dislocation or fragmentation of bone on standard foot radiographs -LSB- 8 -RSB- . All patients had intact feet and no evidence of foot infection or ulceration . Ethical permission for this study was obtained from the King 's College Hospital Research Ethics Committee and all participants gave written informed consent . Isolation and culture of monocytes Peripheral blood mononuclear cells were isolated as previously described -LSB- 6 -RSB- . Briefly , blood was diluted 1:1 in α-minimum essential medium -LRB- MEM ; Invitrogen , Paisley , UK -RRB- , layered over Histopaque and centrifuged -LRB- 693 g -RRB- for 20 min . The interface layer was resuspended in MEM , then centrifuged -LRB- 600 g -RRB- for a further 10 min . The resultant cells were resuspended in MEM with 10 % heat-inactivated FCS and counted in a haemocytometer following lysis of erythrocytes by a 5 % -LRB- vol . / vol . -RRB- acetic acid solution . To assess the extent of osteoclast formation and resorption , PBMCs were cultured on glass coverslips and dentine slices . Initially , 5 × 105 PBMCs were added to 6-mm diameter glass coverslips and 4-mm diameter dentine slices in MEM containing 100 UI/ml penicillin , 100 μg/ml streptomycin and 10 % FCS -LRB- Gibco , Paisley , UK -RRB- . After 2 h incubation , coverslips and dentine slices were vigorously rinsed in medium to remove non-adherent cells . The cultures were maintained in MEM/FCS under three different culture conditions : -LRB- 1 -RRB- human M-CSF -LRB- R&D Systems Europe , Abingdon , UK -RRB- alone at 25 ng/ml ; -LRB- 2 -RRB- M-CSF plus 100 ng/ml human soluble RANKL -LRB- sRANKL ; Peprotech , London , UK -RRB- -LRB- a concentration known to facilitate differentiation of osteoclast precursors to active bone-resorbing osteoclasts in vitro -RRB- ; and -LRB- 3 -RRB- M-CSF plus sRANKL plus 250 ng/ml human OPG -LRB- R&D Systems Europe -RRB- . Coverslips and dentine slices were cultured at 37 °C in 5 % CO2 for 14 and 21 days respectively . Osteoclast formation After 14 days , the coverslips were examined histochemically for the expression of tartrate-resistant acid phosphatase -LRB- TRAcP -RRB- , an osteoclast marker . Coverslips with newly formed osteoclasts were collected and rinsed in PBS buffer , fixed with formalin -LRB- 10 % -LSB- vol . / vol . -RSB- in PBS buffer -RRB- for 10 min and rinsed in distilled water . TRAcP was histochemically revealed by a simultaneous coupling reaction using Naphtol AS-BI-phosphate as substrate and Fast violet B as the diazonium salt . The coverslips were incubated for 90 min at 37 °C in a dark room , rinsed three times in distilled water and the residual activity was inhibited by 4 % NaF -LRB- wt/wt -RRB- for 30 min . Coverslips were then rinsed in distilled water , counterstained with DAPI for 20 min and allowed to dry before mounting , using an aqueous medium . TRAcP-positive cells with more than three nuclei were identified as osteoclasts . The number of newly generated osteoclasts was assessed using a light microscope examination . Osteoclast resorption After 21 days , the dentine slices were removed from the culture wells , placed in NH4OH -LRB- 1 mol/l -RRB- for 30 min and sonicated for 5 min to remove any adherent cells . They were then rinsed in distilled water and stained with 0.5 % -LRB- vol . / vol . -RRB- toluidine blue prior to examination by light microscopy . The surface of each dentine slice was examined for evidence of lacunar resorption and the extent of eroded surface on each dentine slice was determined using image analysis and expressed as the percentage of surface area resorbed . Statistical analyses Data were expressed as a mean ± SEM . Initially the difference within the three study groups -LRB- Charcot patients , healthy and diabetic controls -RRB- was assessed with the non-parametric Kruskall -- Wallis test . Then the differences between Charcot and diabetic patients , and Charcot patients and healthy controls were assessed by the non-parametric Mann -- Whitney U test . In each patient group , the differences between the various culture conditions were also assessed using the Mann -- Whitney U test . Differences were considered significant at p < 0.05 . Results Osteoclast cultures in the presence of M-CSF Osteoclast formation The mean number of newly formed TRAcP-positive multinucleated osteoclasts in the presence of M-CSF alone was significantly greater in the patients with acute Charcot 's osteoarthropathy -LRB- 48.6 ± 18.2 -RRB- than in diabetic -LRB- 6.8 ± 2.7 -RRB- and healthy control participants -LRB- 5.0 ± 0.7 -RRB- -LRB- p = 0.008 -RRB- . The number of TRAcP-positive multinucleated osteoclasts formed in acute Charcot 's osteoarthropathy was 7.2 and 9.7 times greater than those formed in diabetic -LRB- p = 0.010 -RRB- and healthy control groups -LRB- p = 0.003 -RRB- , respectively . Osteoclast resorption The newly formed osteoclasts exhibited increased functional activity as demonstrated by the extent of resorption on dentine slices , with percentage area resorption significantly elevated in the patients with acute Charcot 's osteoarthropathy -LRB- 0.264 ± 0.06 % -RRB- compared with diabetic -LRB- 0.000 ± 0.00 % -RRB- and healthy control groups -LRB- 0.004 ± 0.01 -RRB- -LRB- p < 0.0001 -RRB- . The percentage of resorption was significantly greater in the Charcot patients than in the diabetic -LRB- p = 0.001 -RRB- and healthy control groups -LRB- p = 0.001 -RRB- . Osteoclast cultures in the presence of M-CSF and sRANKL Osteoclast formation The addition of sRANKL led to an increase in the number of TRAcP-positive multinucleated osteoclasts in all three groups of patients . The mean number of these osteoclasts in patients with acute Charcot 's osteoarthropathy was 96.0 ± 21.6 , which was significantly greater than that in the diabetic -LRB- 56.5 ± 11.5 -RRB- and healthy -LRB- 29.0 ± 5.1 -RRB- control groups -LRB- p = 0.010 ; Fig. 1a , c , e -RRB- . The number of TRAcP-positive multinucleated osteoclasts in the patients with acute Charcot 's osteoarthropathy was 1.7 times higher than in diabetic control patients , but this finding did not reach significance -LRB- p = 0.105 -RRB- . However , the number of these osteoclasts in the acute Charcot group was 3.3 times -LRB- and significantly -RRB- higher than in the healthy control group -LRB- p = 0.005 -RRB- . When the number of cells in the cultures with M-CSF alone was compared with that after the addition of sRANKL , there was a significant increase in the diabetic control patients -LRB- from 6.8 ± 2.7 to 56.5 ± 11.5 , p = 0.003 -RRB- and in the healthy participants -LRB- from 5.0 ± 0.7 to 29.0 ± 5.1 , p = 0.002 -RRB- , while the increase in the number of TRAcP-positive multinucleated osteoclasts in the acute Charcot group failed to reach significance -LRB- increase from 48.6 ± 18.2 to 96.0 ± 21.6 , p = 0.059 ; Fig. 2a -RRB- . Fig. 1Multinucleated TRAcP-positive cells were formed on glass coverslips -LRB- a , c , e -RRB- capable of lacunar resorption -LRB- b , d , f -RRB- after 14 and 21 days incubation , respectively , in the presence of 25 ng/ml human M-CSF and 100 ng/ml sRANKL . Newly formed osteoclasts were numerous and highly active in Charcot 's patients -LRB- a , b -RRB- compared with diabetic -LRB- c , d -RRB- and healthy control -LRB- e , f -RRB- participants . Scale bars , 10 μmFig . 2a Quantitative comparison between the number -LRB- n -RRB- of TRAcP-positive cells formed in cultures with M-CSF alone -LRB- white bars -RRB- or with M-CSF and sRANKL -LRB- black bars -RRB- in patients with Charcot 's osteoarthropathy and diabetic and healthy control participants . b Quantitative comparison between the percentage area resorption in the same cultures and patient groups . Statistical differences between the groups were determined using the Mann -- Whitney U test , with significance as follows : a Charcot 's p = 0.059 , diabetic control p = 0.003 , healthy control p = 0.002 ; b Charcot 's p < 0.0001 , diabetic control p < 0.0001 , healthy control p < 0.0001 Osteoclast resorption The percentage area resorption on dentine slices with M-CSF and sRANKL was significantly increased in the acute Charcot group -LRB- 41.6 ± 8.1 % -RRB- compared with that in the diabetic -LRB- 14.2 ± 16.5 % -RRB- and healthy control groups -LRB- 10.5 ± 1.9 % ; p = 0.005 -RRB- . Resorption in the Charcot patients was 2.9 times higher than in diabetic control patients -LRB- p = 0.008 -RRB- and four times higher than in healthy participants -LRB- p = 0.005 ; Fig. 1b , d , f -RRB- . The addition of sRANKL to the cultures with M-CSF led to the following rises in the percentage area resorption when compared with M-CSF alone : Charcot 's 0.264 ± 0.06 % to 41.6 ± 8.1 % , p < 0.0001 ; diabetic control 0.000 ± 0.00 % to 14.2 ± 16.5 % , p < 0.0001 ; healthy control 0.004 ± 0.01 % to 10.5 ± 1.9 % , p < 0.0001 -LRB- Fig. 2b -RRB- . Osteoclast cultures in the presence of M-CSF , sRANKL and excess concentrations of OPG Osteoclast formation The addition of excess concentrations of OPG led to a reduction in the number of TRAcP-positive multinucleated osteoclasts in the cultures with M-CSF , sRANKL and OPG in all the three groups of patients . However , after the addition of OPG , the number of TRAcP-positive multinucleated osteoclasts was still significantly increased in the Charcot group -LRB- 54.4 ± 17.6 -RRB- , as compared with diabetic -LRB- 8.8 ± 5.3 -RRB- and healthy control participants -LRB- 4.4 ± 1.2 ; p = 0.003 -RRB- . In the cultures with M-CSF , sRANKL and OPG , the number of TRAcP-positive multinucleated osteoclasts was greater in the Charcot patients than in the diabetic -LRB- p = 0.005 -RRB- and healthy control groups -LRB- p = 0.001 -RRB- . When OPG was added to the cultures with M-CSF and sRANKL , the reduction in the number of TRAcP-positive cells in Charcot patients was not significant -LRB- 96.0 ± 21.6 versus 54.4 ± 17.6 , p = 0.189 -RRB- . OPG on the other hand significantly inhibited the number of TRAcP-positive cells in M-CSF and RANKL-mediated cultures from diabetic -LRB- reduced from 56.5 ± 11.5 to 8.8 ± 5.3 , p = 0.005 -RRB- and healthy control participants -LRB- 29.0 ± 5.1 to 4.4 ± 1.2 , p = 0.003 ; Fig. 3a -RRB- . Fig. 3a Comparison between the number -LRB- n -RRB- of TRAcP-positive cells formed in cultures with M-CSF and sRANKL -LRB- black bars -RRB- or with M-CSF , sRANKL and excess concentrations of OPG -LRB- 250 ng/ml -RRB- -LRB- grey bars -RRB- in patients with Charcot 's osteoarthropathy and diabetic and healthy control participants . b Comparison between the percentage area resorption in the same cultures and patient groups . Statistical differences between the groups were determined using the Mann -- Whitney U test , with significance as follows : a Charcot 's p = 0.189 , diabetic control p = 0.005 , healthy control p = 0.003 ; b Charcot 's p = 0.001 , diabetic control p = 0.001 , healthy control p < 0.0001 Osteoclast resorption The addition of OPG led to a marked reduction of the percentage area resorption on dentine slices in Charcot patients -LRB- from 41.6 ± 8.1 % to 5.9 ± 2.4 % , p = 0.001 -RRB- and also in diabetic -LRB- 14.2 ± 16.5 % to 0.45 ± 0.31 % , p = 0.001 -RRB- and healthy control -LRB- from 10.5 ± 1.9 % to 0.00 ± 0.00 % , p < 0.0001 -RRB- participants -LRB- Fig. 3b -RRB- . However , the percentage area resorption on the dentine slices was still greater in the cultures with M-CSF , RANKL and OPG from the patients with acute Charcot 's osteoarthropathy -LRB- 5.9 ± 2.4 % -RRB- than in those from diabetic -LRB- 0.45 ± 0.31 % -RRB- and healthy control -LRB- 0.00 ± 0.00 % -RRB- participants -LRB- p = 0.003 -RRB- . Resorption on the dentine slices was greater in the Charcot patients than in diabetic -LRB- p = 0.005 -RRB- and healthy control -LRB- p = 0.003 -RRB- groups . Discussion This study shows that monocytes from patients with acute Charcot 's osteoarthropathy cultured in the presence of M-CSF alone were capable of differentiating into mature osteoclasts that exhibited increased resorption compared with diabetic and healthy control participants . Furthermore , osteoclasts generated after the addition of sRANKL were functionally more aggressive , exhibiting a considerable increase in the extent of resorbing activity in patients with acute Charcot 's osteoarthropathy . This resorption was partially blocked by the addition of excess concentrations of OPG , a soluble receptor decoy for RANKL . This suggests that the increased osteoclastic activity in patients with acute Charcot 's osteoarthropathy is mediated through both a RANKL-dependent and a RANKL-independent pathway . Cultures from the patients with Charcot 's osteoarthropathy showed increased osteoclast formation and resorption when cultured with M-CSF alone . Although M-CSF is an essential factor for proliferation , differentiation and survival of the monocyte-macrophage lineage -LSB- 9 , 10 -RSB- , it is not an osteoclastogenic factor and it is unusual to detect osteoclast formation and resorption in cultures with M-CSF , as was seen in the diabetic and healthy controls . This observation suggests that in acute Charcot 's osteoarthropathy there may be increased levels of other circulating pro-inflammatory factors such as TNF-α -LSB- 11 , 12 -RSB- , IL-6 -LSB- 13 -RSB- , IL-8 -LSB- 14 -RSB- and LIGHT -LRB- homologous to lymphotoxins exhibiting inducible expression and competing with herpes simplex virus glycoprotein D for herpes virus entry mediator -LSB- HVEM -RSB- , a receptor expressed by T lymphocytes -RRB- -LSB- 15 -RSB- , which have been previously shown to stimulate osteoclastogenesis independently of RANK/RANKL mechanisms . The concentrations of these circulating factors in diabetic and healthy control participants may not be sufficient to induce the formation and differentiation of active osteoclasts in the presence of M-CSF alone . After the addition of sRANKL to M-CSF cultures , the newly formed osteoclasts exhibited markedly increased resorption in the patients with Charcot 's osteoarthropathy , although the number of osteoclasts did not significantly increase in these patients compared with cultures with M-CSF alone . These observations may not be unique to Charcot 's osteoarthropathy , and indeed similar observations have been reported in other conditions associated with increased bone resorption , such as rheumatoid arthritis where the addition of sRANKL resulted in a significant increase in lacunar resorption , but did not lead to a significant increase in the number of TRAcP-positive cells -LSB- 16 -RSB- . Overall , the observed extensive resorption in acute Charcot patients , in the presence of M-CSF and sRANKL , as compared with the diabetic or healthy control groups , may suggest that the osteoclast precursors circulating in acute Charcot patients are in a higher activated state and as such are more primed to becoming osteoclasts -LRB- mediated through RANKL -RRB- than those in the control groups . In order to ascertain that RANKL was a major osteoclastic activator in patients with Charcot 's osteoarthropathy , excess concentrations of OPG , the soluble receptor decoy to RANKL , were added to the cultures with M-CSF and RANKL . The rationale for this approach was that if osteoclastogenesis is mediated solely through RANK -- RANKL interaction , addition of excess concentrations of OPG -LRB- as had been previously determined to be sufficient to block osteoclastogenesis through RANKL -LSB- 15 -RSB- -RRB- would completely abolish the process of osteoclast differentiation and activation . In the current study , although osteoclast formation and resorption in the diabetic and healthy control groups was completely blocked by the addition of OPG , the latter did not achieve total inhibition of osteoclast formation and resorption in patients with acute Charcot 's osteoarthropathy . These results suggest that although RANKL-dependent pathways do play a significant role in the osteoclastic activity of Charcot 's osteoarthropathy , an alternative pathway -LRB- other than RANK/RANKL -RRB- may also be involved . Osteoclastogenic mediators other than RANKL that have been reported to stimulate osteoclast differentiation independently of the RANKL pathway include TNF-α -LSB- 11 , 12 -RSB- , IL-6 -LSB- 13 -RSB- , IL-8 -LSB- 14 -RSB- and LIGHT -LSB- 15 -RSB- . In acute Charcot 's osteoarthropathy , it is possible that one or a combination of these factors may have initiated the circulating osteoclast precursors to be in a more ` primed ' condition , a situation which as such could explain the observed resorption in Charcot monocyte cultures supplemented with M-CSF alone , without the exogenous addition of any osteoclastogenic mediators . The osteolysis of Charcot 's osteoarthropathy may be explained by our observation that osteoclast precursors from Charcot patients develop into mature osteoclasts that exhibit increased resorptive activity , especially in response to RANKL , unlike the increased resorption in response to bacterial infection , which is not mediated by RANKL -LSB- 17 -RSB- . Increased expression of RANKL has been previously demonstrated in pathological osteolysis associated with the development of various bone diseases -LSB- 18 -RSB- and a similar mechanism may contribute to osteolysis of Charcot 's osteoarthropathy -LSB- 5 -RSB- . Furthermore , patients with Charcot 's osteoarthropathy have severe neuropathy , which itself can also lead to increased expression of RANKL as a result of the loss of nerve-derived peptides known to antagonise its effect such as calcitonin gene-related peptide -LSB- 5 -RSB- . In addition to the RANKL-dependent pathway , our results suggest that a RANKL-independent pathway , mediated by pro-inflammatory cytokines , may also be important . Indeed , Charcot 's foot is characterised by excessive inflammation and proinflammatory cytokines have been implicated in its pathogenesis -LSB- 19 -RSB- . In support of this , a recent immunohistochemical analysis of bone samples isolated from Charcot 's osteoarthropathy patients showed excessive osteoclastic activity in a microenvironment enriched with mediators of bone resorption -LRB- IL-1 , IL-6 and TNF-α -RRB- -LSB- 20 -RSB- . Thus a RANKL-independent pathway , which is also known to play a role in other osteolytic disorders such as rheumatoid arthritis -LSB- 21 -RSB- and aseptic loosening -LSB- 22 -RSB- , could contribute also to the pathogenesis of the Charcot 's osteoarthropathy . This study has indicated , for the first time that the RANKL-dependent pathway is important in the pathogenesis of Charcot 's osteoarthropathy , thereby raising the possibility of the use of RANKL inhibition in the management of Charcot 's foot . However , our observations also suggest that a RANKL-independent pathway may play a role , but further investigation is required to fully clarify the mechanism involved . If confirmed , specific pharmacological agents that counteract the RANKL-independent pathway , such as anti-TNF strategies , may be useful in the treatment of Charcot 's osteoarthropathy . Whatever the relative importance of either pathway , this in vitro technique of generating human osteoclasts from PBMCs may allow specific characterisation of osteoclastic activity in each patient and could , in the future , lead to individually tailored anti-osteoclastic treatment for the patient with acute Charcot 's osteoarthropathy .

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opg osteoclasts osteolysis rankl resorption