Aims: To investigate whether plasma biomarkers for axonal injury and inflammation are related to loss and recovery of visual function in acute optic neuritis (ON).Methods: Eighteen patients with ON and 14 controls were investigated in a longitudinal, prospective study. Plasma phosphorylated neurofilament heavy chain ( NfH(SMI35); a surrogate marker of axonal injury), nitric oxide metabolites (NOx), and citrulline ( surrogate markers of inflammation) were measured.Results: Patients with ON had higher median plasma NfH(SMI35) values than controls (0.17 versus 0.005 ng/ml; p< 0.05) and higher NOx values (49 versus 35.5 mu M; p< 0.001). Plasma NfH(SMI35) values correlated inversely with visual acuity at presentation ( R = -0.67; p = 0.01). NfH(SMI35) was higher in patients with poor recovery of visual acuity than in those with good recovery (0.25 ng/ml versus 0.09 ng/ml; p< 0.05). Three of four patients with high NfH(SMI35) and high NOx values experienced a poor recovery as opposed to only one of five with high NOx but normal NfH(SMI35) values.Conclusions: NfH(SMI35), a surrogate marker for axonal damage, is a prognostic indicator and should be considered in the design of neuroprotective treatment strategies

Petzold, A

Plant, GT

Rejdak, K

English

PubMed

A Petzold

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Axonal degeneration and inflammation in acute optic neuritis

UCL Discovery

Axonal degeneration and inﬂammation in acuteoptic neuritisA. Petzold a;c; K. Rejdak a;d G.T. Plant b;caUCL Institute of Neurology, Department of Neuroinﬂammation, University CollegeLondon, Queen Square, London WC1N 3BG, United KingdombNational Hospital for Neurology and Neurosurgery, Queen Square, London,WC1N 3BG, UKcMoorﬁelds Eye Hospital, Department of Neuro–ophthalmology, City Road, EC12PD, UKdDepartment of Neurology, University of Lublin, PolandPreprint submitted to JNNP 01.09.2003AbstractThis longitudinal study of acute optic neuritis (ON) investigated whether plasmabiomarkers for axonal injury and inﬂammation could be related to loss and recoveryof visual function.Eighteen patients with ON and 14 control patients were included into this prospec-tive study. Plasma NfHSMI35 (a surrogate for axonal injury), NOx and citrulline (sur-rogates for inﬂammation) were measured by ELISA.Patients with ON had higher median plasma NfHSMI35 levels when comparedto controls (0.17 ng/mL versus 0.005 ng/mL, p<0.05) and higher NOx levels (49M versus 35.5, p<0.001). Plasma NfHSMI35 levels correlated inversely with visualacuity at presentation (R=-0.67, p=0.01). NfHSMI35 was higher in patients with poor(0.25 ng/mL) when compared to those with good recovery of visual acuity (0.09ng/mL, p<0.05). Seventy–ﬁve percent of patients with high NfHSMI35 and high NOxlevels experienced a poor recovery as opposed to only 20% with high NOx butnormal NfHSMI35 levels.NfHSMI35 a surrogate marker for axonal damage is a prognostic indicator andshould be considered in the design of neuroprotective treatment strategies.Key words: Surrogate marker, NfHSMI35 , NOx, citrulline, ON, axonal loss Department of Neuroinﬂammation, Institute of Neurology, Queen Square, Lon-don WC1N 3BG, United Kingdom. Tel.: +44 207 837 3611 ext. 4204, Fax +44 207837 8553, Email: a.petzold@ion.ucl.ac.uk2The “axonal death cascade” (1) has been associated to nitric oxide (NO)mediated damage in vitro (2). The relation of axonal degeneration in vivo toNO release is not known. Acute optic neuritis provides a useful model be-cause of the deﬁned onset and the anatomically restricted area of neuronaldamage (3; 4). Increased levels of the NO metabolites nitrite (NO2 ) andnitrate (NO3 ) were found in the cerebrospinal ﬂuid and serum of patientswith optic neuritis (5; 6). Axonal loss appears to be linked to the acquisitionof permanent deﬁcit (7; 2; 8; 9; 10; 11; 12). However the role of inﬂamma-tion in demyelinating disease is less well deﬁned and there is accumulatingevidence that immune-mediated inﬂammation possesses neuroprotectiveproperties (1; 13).This study aimed to investigate the relationship between loss and recoveryof optic nerve function with surrogate markers for inﬂammation (citrullineand the NO metabolites NO2  and NO3  (14; 15; 2; 16)) and for axonalinjury (neuroﬁlaments (17)) in patients with acute optic neuritis.1 Patients and MethodsPatients presenting with symptoms suggestive of acute unilateral optic neu-ritis (n=18) and an age-matched healthy control group (n=14) were includedin this prospective study (Table 1). Inclusion criteria were: progressive lossof vision over a few days, decreased visual acuity (Snellen chart), decreasedcolour vision (Ishihara colour plates) and relative afferent pupillary defect(3). Because visual acuity (VA) was in most cases severely affected thevisual ﬁelds (VF) were usually assessed by confrontation. As this is not aquantitative method this precluded the use of perimetry in the quantitativeassessment. Exclusion criteria were: any other macula or retinal pathology,contemporaneous symptoms suggestive demyelination elseswere or lack ofspontaneous recovery. The study was approved by the local ethics commit-tee. Recovery of VA and colour vision was assessed at each visit and oncerecovery plateaued no further follow-up was arranged. The last recorded VAwas taken as the ﬁnal outcome. Brain and optic nerve MR imaging was per-formed using a 1.5 Tesla system (Siemens AG, Erlangen, Germany) andconsisted of an axial T1 and T2 weighted spin echo MR imaging (3; 5).Plasma was taken at the ﬁrst visit and levels of the phosphorylated neu-roﬁlament heavy chain (NfHSMI35 ), NOx and citrulline were measured asdescribed (17; 16; 14).Data analysis was performed using SAS. Because of non-Gaussian datadistribution the median and interquartile range are shown. All correlationswere studied using Spearman rank correlation coefﬁcient. Differences be-tween groups were compared using the two-sided Wilcoxon two-sample3test. Signiﬁcances based on small numbers were checked on a categoricallevel using the two–sided Fischer’s exact test. Trend analysis was using theMantel-Haenzel (M-H2) test. A 5 % level of signiﬁcance was used through-out.2 ResultsThe diagnosis of optic neuritis was conﬁrmed in all 18 patients during follow-up. However in 4 patients the blood sample was taken over 4 weeks afteronset of symptoms. These patients were therefore excluded from analysis.The remaining 14 patients presented with a median time from onset of 2weeks (Table 1). At presentation 10/14 (71%) of the patients had a VA ofless then 0.33 (6/18, 20/60 Snellen equivalent). In total 13/14 (93%) of pa-tients showed sustained improvement of their VA during follow-up. A minordegree of optic atrophy was observed in 44% of patients whose VA recov-ered to over 0.33 (6/18). Optic nerve atrophy was more severe in those 36%of patients who did not recover their VA above 0.33.Plasma levels of NfHSMI35 (Figure 1 A) and NO metabolites (NOx) (Figure1 B) were signiﬁcantly higher in ON patients than in controls (p<0.001,p<0.01, respectively). No such difference was found for citrulline (Table 1).There was a negative correlation between NfHSMI35 and VA at the time ofﬁrst presentation (R=-0.67, p=0.01, Figure 1 C). One outlier with NfHSMI35 levelsof 0.65 ng/mL was observed. This 48 old male patient presented with a rightON, VA was reduced to hand movement (6/60, 20/200 Snellen equivalent),colour vision to 0/17 and there was a right RAPD. This patient had sufferedfrom an episode of myelitis 2 years ago. In the absence of any clinical signsof myelitis no spinal MRI was performed. After the removal of this patientfrom the study the correlation between NfHSMI35 and VA remained signiﬁ-cant (R=-0.60, p<0.05), but the slope of the linear regression decreasedfrom -0.22 to -0.145.No correlation between VA and either NOx or citrulline was found at presen-tation. There was no correlation for either NfHSMI35 , NOx or citrulline withage, time from onset and there was no gender difference.Plasma NfHSMI35 levels at presentation were signiﬁcantly higher (0.25 ng/mL)in the 36% of the patients with poor recovery of visual function (0.33, 6/18,20/60 Snellen equivalent) when compared to those whose VA recoveredto above 0.33 (0.09 ng/mL, p<0.05). Signiﬁcance remained comparing pro-portions of patients with NfHSMI35 levels above cut–off (Figure 1 A, p=0.015,two–sided Fisher’s exact test). There was no difference for the NOx levelsat presentation between those with good versus poor visual recovery at4follow-up (54.2 versus 44.6 M, p=0.1).The combined relationship of elevated NOx and NfHSMI35 levels with recov-ery of visual function was investigated by comparing proportions of pa-tients with high levels (elevated above the top value of the control group;NfHSMI35 > 0.17 ng/mL, NOx > 43.8 M). There was a signiﬁcant trend forincreased proportion of patients experiencing a poor recovery (M-H2=4.6,p<0.05) across the following categories: NfH normal and NOx normal (0/3,0% poor recovery), NfH normal and NOx high (1/5, 20% poor recovery),NfH high and NOx normal (1/2, 50% poor recovery), NfH high and NOxhigh (3/4, 75% poor recovery).MRI was available in 10 of 14 patients, 6/10 had an isolated ON lesion(NfHSMI35 0.12 ng/mL, NOx 40.7 M) as opposed to 40% with disseminatedbrain lesions (NfHSMI35 0.17 ng/mL, NOx 52.9 M). Although NfHSMI35 andNOx seemed to be marginally higher in patients with disseminated brainlesions this did not reach statistical signiﬁcance.3 DiscussionThis is the ﬁrst study to measure biomarkers for axonal injury (NfHSMI35 )and inﬂammation (NOx, citrulline) in the plasma of patients with acute opticneuritis. The main ﬁndings were that NfHSMI35 levels were higher in ON thanin control patients, were inversely correlated to visual acuity at presentationand were of prognostic signiﬁcance. This suggests the presence of axonaldamage in the acute phase. The results are in line with clinical, MRI andelectrophysiological evidence for optic nerve ﬁbre loss following a singleepisode of ON and insidiously in MS (18; 19; 20).Recovery of the VA to  6/18 (20/60) was achieved by less patients (36%)compared to VA  20/50 (88%) in the optic neuritis treatment trial (ONTT)after 5 years follow–up (4). Bradley and Whitty (1967) found that recovery ofVA to  20/30 was achieved by 50% after 1 month and 75% after 6 months(21). The result of the present study may relate to the small sample in thisstudy and the relatively short period of follow up (25–189 days).Recovery of VA in MS related ON has been attributed to resolution of con-duction block caused by acute inﬂammation (19; 20). NO mediated con-duction block also seems to be the initial mechanism in in vitro studies(2; 22; 23). The present and one other study (5) failed to show a signiﬁcantrelationship between serum NOx levels and outcome. Additionally NOx andNfHSMI35 did not correlate with MRI parameters, but this could be due to thesmall study population. The almost 30-40% higher values, if true, might in-5dicate demyelinating activity elsewhere contributing to the worse outcome.Citrulline was thought to be a second marker for NO mediated inﬂammation,but it did not correlate with NOx and no difference between ON and controlpatients could be shown. This ﬁnding is in keeping with one study com-paring CSF and serum citrulline levels between patients with Lewy bodydementia and healthy controls (18). We agree with these authors that cit-rulline is not a good surrogate for NO metabolism because it is a substratefor enzymes other than iNOS.In MS it appears that inﬂammatory markers were predominately related todisease activity and the MRI lesion load but not outcome (5; 24). In additionepidemiological data suggested that inﬂammation in MS only has a limitedeffect on the course of neurodegeneration (25). In placebo–controlled trialsand clinical experience with ON anti-inﬂammatory treatment with steroidsdoes not inﬂuence outcome in the majority of cases (3). The destructiveand neuroprotective aspects of inﬂammation have stimulated a controver-sial discussion (1; 13; 26; 25; 5; 27). It is therefore interesting that in thissmall group 4/5 (80%) of patients with elevated NOx levels but normalNfHSMI35 levels made a good recovery. This ﬁnding highlights the potentialfor selection bias in treatment trials for neuroprotective drugs by inclusionof those patients with evidence of inﬂammation only.Taken together, these ﬁndings support the concept that sustained loss ofoptic nerve function relates to axonal degeneration and that NO might atleast in part contribute to the “axonal death cascade” (1; 2; 8; 9; 7).6References[1] S. Waxman, Nitric oxide and the axonal death cascade, Ann Neurol 53(2003) 150–103.[2] R. Kapoor, M. Davies, P. Blaker, S. Hall, K. Smith, Blockers of sodiumand calcium entry protect axons from nitric oxide-mediated degenera-tion, Ann Neurol 53 (2003) 174–180.[3] S. Hickman, C. Dalton, D. Miller, G. Plant, Management of acute opticneuritis, Lancet 360 (2002) 1953–1962.[4] R. Beck, Walsh and Hoyt’s Clinical Neuro–Ophthalmology, 5th Edition,Williams and WIlkins, 1998, Ch. Optic Neuritis, pp. 599–647.[5] G. Giovannoni, D. Miller, N. 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Whitty, Acute optic neuritis: its clinical features and theirrelation to prognosis for recovery of vision., J Neurol Neurosurg Psy-chiatry 30 (6) (1967) 531–508.[22] K. Smith, S. Hall, Factors directly affecting impulse transmission ininﬂammatory demyelinating disease: recent advances in our under-standing, Curr Opin Neurol 14 (2001) 289–298.[23] K. Smith, R. Kapoor, S. Hall, M. Davies, Electrically active axons de-generate when exposed to nitric oxide, Ann Neurol 49 (2001) 470–406.[24] G. Acar, F. Idiman, E. Idiman, et al., Nitric oxide as an activity markerin multiple sclerosis, J Neurol 250 (2003) 588–592.[25] C. Confavreux, S. Vukusic, P. Adeleine, Early clinical predictors andprogression of irreversible disability in multiple sclerosis: an amnesicprocess, Brain 126 (2003) 770–782.[26] C. Confavreux, S. Vukusic, T. Moreau, P. Adeleine, Relapses and pro-gression of disability in multiple sclerosis, N Engl J Med 343 (2000)1430–1438.[27] P. Rieckmann, K. Smith, Multiple sclerosis: more than inﬂammationand demyelination, Trends Neurosci 24 (2001) 435–407.8Table 1: Characteristics of patients expressed as me-dians (interquartile range). MRI = magnetic resonanceimaging, na = not assessed, RAPD = relative afferentpupillary deﬁcit, VFD = visual ﬁeld defect.Feature Controls Acute optic neuritis SigniﬁcanceAge (years) 36 (30–43) 36 (35-46)Male: Female 4:10 9:5 p<0.05Eye N/A R 8 : L 6RAPD N/A R 8 : L 5Visual acuity 1.2 0.2 (0.05 – 0.67)Colour vision 17/17 0/17 (0/17 – 7/17)Disc appearance Normal 2 swollen, 12 normalTime from onset N/A 14 days (8 – 26)Follow–up N/A 171 days (25 – 189)MRI N/A 10Plasma NfHSMI35 (ng/mL) 0.005 (0.0 – 0.094) 0.17 (0.07 – 0.33) p<0.001Plasma NOx (M) 36.2 (31.2 – 38.6) 48.1 (40.8 – 54.2) p<0.01Plasma Citrulline (nmol/mL) 83.4 (72.1 – 92.4) 67.9 (62.5 – 91.9) N.S.9(A)(B)(C)Figure 1. (A) Plasma NfHSMI35 (ng/mL) levels and (B) NOx (M) levels in con-trols (CTRL) and patients with acute optic neuritis (ON). The median (50%), box(25%-75%) and whisker (0%-100%) are shown next to the individual values (opencircle). (C) Plasma NfHSMI35 correlates with visual acuity (VA) at time of sampling(R=-0.66, p=0.01).10

Axonal degeneration and inflammation in acute optic neuritis

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