1. CORRELATION OF BIOMARKERS FOR AXONAL DEGENERATION WITH RETINAL NERVE FIBER LAYER THICKNESS AND DISABILITY IN DIFFERENT PHASES OF MULTIPLE SCLEROSIS
UZUNKOPRU C(*), YUCEYAR N(*), YILMAZ S(**), TASKIRAN D(***), AFRASHI F(**), EKMEKCI O(*)
EGE UNIVERSITY MEDICAL SCHOOL OF HOSPITAL, BORNOVA, IZMIR, TURKEY
(*) DEPARTMENT OF NEUROLOGY, (**) DEPARTMENT OF OPTHALMOLOGY (***) DEPARTMENT OF PYHSIOLOGY
Background: Neurofilaments (Nf) and nitrotyrosine(NT) are promising candidates to be a reliable biomarkers of axonal degeneration in multiple sclerosis (MS).An indirect measuring of peripapillary retinal nerve fiber layer (RNFL) has been also proposed as a way to verify axonal loss. Objectives:   The aim of the study is to confirm the potential of Nf as an early biomarker of axonal degeneration in MS and to assess its relationship with other biomarker; nitrotyrosine (NT)and with retinal nerve fiber thickness(RNFT). We also investigate their correlation to clinical disability and cognitive decline in different phases of MS.   Methods: NF heavy chain (NfH) and NT were analyzed by ELISA in serum and cerebrospinal fluid (CSF) of 30 relapsing remitting MS patients (RRMS)(mean age 30,5±6.1years) fulfilling the McDonald 2010 criteria (in whom 23 patients were evaluated after their first demyelinating event) and in serum of 16 secondary progressive MS (SPMS) patients (mean age 40±6.8years).Blood and CSF samples were collected at least 30 days after their inflammatory attack. 21 healthy subjects and 8 non-inflammatory neurological patients with the diagnosis of pseudotumor cerebri matched for age and gender were served as controls. The thickness of RNFL was measured in both eyes by optical coherence tomography (OCT) in all subjects. Multiple Sclerosis Functional Composite (MSFC), Expanded Disability Status Scale (EDSS) and Trail Making Test were also performed to all subjects. Results: Demographic data: In RRMS patients, the onset of the first demyelinating event was within the last 2 years. SPMS patients with at least 10 years of disease duration were included in the study. The mean EDSS scores were (2,2±0,6) in RRMS and (4,9±0,8) in SPMS.   Neurofilament levels: RRMS patients showed significantly increased CSF neurofilament levels (118,6±81,8) compared with patients with pseudotumor serebri (73,9±17,5) (p<0,002) ( Figure-1) Serum neurofilament levels were also significantly increased in RRMS patients compared to healthy subjects and control patients with non-inflammatory neurological disease. (p≤0,001) (Figure-2) SPMS patients exhibited significantly incresed serum neurofilament levels (87,2±18,5) compared with healthy controls (61,8±13,2), with RRMS patients (71,7±16,3) and also with control patiens with pseudotumor serebri (59,0±11,7) (p≤0,05) (Figure-2 ) Nitrotyrosine levels: RRMS patients had significantly higher levels of CSF nitrotyrosine levels (123,6±79,8), compared with control patients with pseudotumor cerebri (65,8±38,5) (p≤0,001) (Figure-3) Serum nitrotyrosine levels were significantly higher than healthy controls in RRMS ( Figure 4) In SPMS, serum nitrotyrosine levels were significantly increased(1158±239) when compared to both RRMS (985±281), healthy controls (732±244) and also with patients with pseudo dotumör cerebri(991±124) (p≤0,05).(Figure 4) OCT Findings: Irrespective of optic neuritis history, all MS patients exhibited significantly lower RNFL thickness of each eyes compared with controls (p<0,05). (Figure 5) The mean ratio of RNFL thickness of both eyes were also significantly reduced in RRMs and SPMS when compared with controls. In SPMS the NLF thickness of each eyes was significantly reduced than patients with RRMS. There was no significant difference between controls with respect to OCT findings. (Figure 5) Disease duration of MS patients correlated significantly with the levels of Nf, NT and thickness of RTNL Correlation of serum and CSF biomarkers with RNFL RNFL thickness was significantly correlated with serum and CSF Nf/NT levels both in RRMS and SPMS patients except for CSF NT levels in RRMS (p<0,05).Table-1, Table-2 Disability and cognitive decline in MS patients All MS patients had significantly lower scores of PASAT and Trail making test than control patients. The difference between SPMS and RRMS patients were also significant. Correlation of RNFL thickness with disability In RRMS patients, RNFL thickness of both eyes was negatively correlated with EDSS and Trail making test scores and positively correlated with PASAT scores. In SPMS patients negative correlation was found between RNFL thickness and EDSS , trail making test, 25 foot walking test scores . Positive correlation was also found between RNFL thickness and PASAT scores. Table-3 Correlation of serum/CSF biomarkers with disability In RRMS only the Nf levels both in sera and CSF were correlated positively with EDSS and Trail making test scores .In SPMS patient only a positive correlation was found between serum NT and Nf levels with EDSS. No correlation was found between biomarkers and PASAT, 24 foot test and nine hole peg test. MSFC scores was significantly negatively correlated with serum NT and Nfl levels , but not with CSF levels . A positive correlation was found between MSFC and RNFL thickness of both eyes. Table-4
Conclusion Our study confirmed that axonal degeneration occurs at the very early stage of MS. The primary findings of this comparative study is that RNFL , serum/ CSF Nf and NT are reliable and quantitative markers for axonal damage and neurodegeneration reflecting different phases of MS. They all have discriminatory power to distinguish RRMS and SPMS. Measurement of RNFL as a noninvasive technique correlated strongly with Nf and NT the other biological markers for axonal degeneration. A strong correlation was found with OCT findings and the clinical disability in all stages of MS while this association for NT and Nf levels was relatively weak.. Only serum and CSF Nf levels correlated well with EDSS scores in RRMS. A correlation between sera of NT and NF levels and EDSS scores was significant in SPMS patient. OCT findings were also found to be have more superior to NT and Nf for distinguish cognitive decline in all stages of MS while this association was not absent for NT and Nf . Our study support the role of RNFL and serum biomarkers of NT and Nf as a useful measure of disability and their potential usefulness as a surrogate measure in MS treatment studies.
Figure-2 CSF nitrotyrosine levels in RRMS and controls
Figure-3 Serum levels of neurofilament in MS patients and controls
Figure-1 CSF neurofilament levels in RRMS and controls
Figure-4 Serum levels of nitrotyrosine in MS patients and controls
Table-3 Correlation of RNFL thickness with physical and cognitive disability
EDSS
Trail Making Test
25 Foot Walking Test
9 Hole Peg Test
PASAT
RRMS
Right eye OCT r
-0,361
-0,194
0,013
0,158
0,267 p
0,034
0,078
0,946
0,403
0,154
Left eye OCT
-0,549
-0,234
0,120
0,195
0,339
0,002
0,033
0,527
0,301
Median OCT right-left eye
-0,378
-0,235
0,069
0,194
0,332
0,040
0,716
0,304
SPMS
-0,543
-0,432
0,128
0,175
0,340
0,020
0,103
0,356
-0,426
-0,552
-0,344
0,275
0,333
0,001
0,187
Median OCT right-left eye
-0,485
-0,357
-0,401
0,534
0,374
0,003
0,000
Pearson correlation
Figure- 5 Pathological OCT findings in any eyes in MS patients
with comparision to controls
Table-2 Correlation of serum neurofilament/nitrotyrosine levels with OCT parameters in SPMS patients
Table-1 Correlation of serum and CSF neurofilament/nitrotyrosine levels with OCT parameters in RRMS patients
Table-4 Correlation of MSFC scores to CSF/serum Nf/NT levels and to RNFL thickness
Neurofilament CSF
Neurofilament Serum
Nitrotyrosine CSF
Nitrotyrosine Serum
MSCF Score r
0,159
-0,459
0,017
-0,361 p
0,339
0,000
0,920
0,001
Right eye OCT
Left eye OCT
Median OCT right-left eye
0,482
0,534
0,541
Pearson correlation
Nf CSF
Nf Serum
NT CSF
NT Serum
RRMS
Right eye OCT r
-0,265
-0,340
0,042
-0,222 p
0,040
0,000
0,824
0,056
Left eye OCT
-0,110
-0,290
0,114
-0,311
0,020
0,549
Median OCT right-left eye
-0,213
-0,350
0,084
-0,291
0,660
Pearson correlation
Neurofilament Serum
Nitrotyrosine
Serum
SPMS
Right eye OCT r
-0,378
-0,234 p
0,000
0,058
Left eye OCT
-0,341
-0,371
0,020
Median OCT right-left eye
-0,360
-0,380