1. Methods, Results and Limitations of Studies of Brain Iron in RLS in the Living Christopher J Earley MB, BCh, PhD, FRCP(I) Professor Department of Neurology Johns Hopkins School of Medicine Christopher J Earley MB, BCh, PhD, FRCP(I) Professor Department of Neurology Johns Hopkins School of Medicine

2. Nordlander (1954), based on his results with IV iron therapy in RLS without ID, stated: “it is possible…that there can exist an iron deficiency in the tissues in spite of normal serum iron”

3. Cerebrospinal Fluid Earley 2000; Mizuno 2005; Earley 2005; Clardy 2006 Cerebrospinal Fluid Earley 2000; Mizuno 2005; Earley 2005; Clardy 2006

4. Serum Iron, Ferritin and Transferrin in Control and RLS Subjects Earley et al, 2000

5. CSF Iron, Ferritin and Transferrin in Control and RLS Subjects Earley et al, 2000

6. Correlation between CSF and Serum Ferritin in Controls vs RLS Subjects Earley et al, 2000

7. Correlation between CSF and Serum Ferritin in Controls vs RLS Subjects Mizuno et al, 2005 Control RLS

8. Correlation between CSF Ferritin and Age of RLS Symptom Onset Earley et al, 2005 r= 0.64, p<0.0002

9. Circadian changes in CSF ferritin in RLS and control subjects Earley et al, 2005 RLS Control

10. Mean (± SEM) CSF Ferritin for Controls and for Early- and Late-onset RLS 0.5 1 1.5 2 2.5 3 3.5 CSF Ferritin (mcg/l ) Controls Early-onset Late- onset (22) (15) RLS (15) Controls Early-onset Late- onset (22) (15) RLS (15) F=5.4, p<0.008 Earley et al, 2005

11. Mean (± SEM) CSF Ferritin for Controls and for Early- and Late-onset RLS with matched age ranges (50-75) in each group 0.5 1 1.5 2 2.5 3 3.5 CSF ferritin (mcg/l) Controls Early-onset Late-onset (17) (12) RLS (10) Controls Early-onset Late-onset (17) (12) RLS (10) F=5.1, p<0.012 Earley et al, 2005

12. CSF Pro-Hepcidin in Controls and in Early-onset and Late-onset RLS Subjects Clardy et al, 2006

13. Brain Iron Imaging

14. Magnetic Resonance Imaging (MRI) Allen 2001 Earley 2006 Lee 2007 Godau 2008 Astrakas 2008 Allen 2001 Earley 2006 Lee 2007 Godau 2008 Astrakas 2008

15. MRI-Determination of Iron  The MR parameters that correlate to tissue iron levels are the transverse relaxation rates (i.e., inverse of relaxation times(T2)) R2, R2*, and R2'.  R2 from spin-echo MRI sequence (1/T2) is the irreversible transverse relaxation rate  R2* from the gradient–echo MRI (1/T2* )  R2' = R2*- R2 due to field inhomogeneties that can be reversed by an 180˚ pulse.  The MR parameters that correlate to tissue iron levels are the transverse relaxation rates (i.e., inverse of relaxation times(T2)) R2, R2*, and R2'.  R2 from spin-echo MRI sequence (1/T2) is the irreversible transverse relaxation rate  R2* from the gradient–echo MRI (1/T2* )  R2' = R2*- R2 due to field inhomogeneties that can be reversed by an 180˚ pulse.

16. MRI-Determination of Iron  R2 proportional to the local iron concentration  R2 and R2* altered by changes in water content or partial volume with CSF (the lower the iron, the higher the water effect)  R2’ is solely affected by local field inhomogeneities  The iron core of ferritin form local microscopic magnetic field inhomogeneities. Other forms of iron have little effect on either R2 or R2’.  R2 proportional to the local iron concentration  R2 and R2* altered by changes in water content or partial volume with CSF (the lower the iron, the higher the water effect)  R2’ is solely affected by local field inhomogeneities  The iron core of ferritin form local microscopic magnetic field inhomogeneities. Other forms of iron have little effect on either R2 or R2’.

17. R2 and R2’ values versus non-heme iron concentrations [Fe] in different brain regions

18. T2* MRI Relaxation Sequence of the Midbrain ( substantia nigra and red nucleus). T2* (sec -1 ) 30 0 RLS Normal

19. Summary of the MRI Studies in RLS Subjects Author year N subjects Change SN Change Other Areas MRI Measure MRI iron Index and Associations Allen 2001 C = 5 R = 5 yes↓ PU 1.5 tesla R2’ (GESFIDE) JHRLSS/SN r 2 = -0.48 Earley 2006 C = 39 (E) = 22 (L) =19 (E) Yes (L) No (L) ↑ PU, PN 3.0 tesla R2’ JHRLSS/SN r 2 = -0.1 none Lee 2007 C = 16 R = 16 yes ↓ RN,TH,PU, GP 1.5 tesla R2,voxel IRLSS/SN r 2 = -0.41 Godau 2008 C =19 R = 6 no↓ TH, CA 1.5 tesla R2 SN Echogenicity /Overall MRI r 2 = -0.37 Astrakas 2008 C = 12 (L) = 25 Yes (SNc) no 1.5 tesla R2 No correlation to JHRLS or IRLSS

20. Substantia Nigra iron content as determined by MRI R2' value RLS Control MRI R2 '

21. Correlation between MRI-R2 value of the substantia nigra and RLS severity Lee et al, 2007

22. MRI-Determined Iron in Several Brain Regions in Control and in Early- and Late-onset RLS

23. Comparison of T2 Relaxation Time (µsec) between RLS and Controls for Several Brain Regions

24. Transcranial Ultrasound Schmidauer 2005 Zecca 2005 Godau 2007, 2008 Schmidauer 2005 Zecca 2005 Godau 2007, 2008

25. Transcranial Ultrasound of Midbrain in a Parkinson’s, normal and RLS subject Transcranial Ultrasound of Midbrain in a Parkinson’s, normal and RLS subject Schmidauer et al, 2005

26. Concentration of Iron in the SN vs Area of Hyperechogenicity in the Postmortem SN Zecca et al, 2005r = 0.3, p = 0.006

27. Concentration of H-ferritin in the SN vs Area of Hyperechogenicity in the Postmortem SN Zecca et al, 2005r = 0.27, p = 0.016

28. Concentration of L-ferritin in the SN vs Area of Hyperechogenicity in the Postmortem SN Zecca et al, 2005r = 0.24, p = 0.03

29. Box Plot of the Total Area of Hyper echogenicity in the SN of Controls (20), Parkinson's (20) and RLS (20) Subjects Schmidauer et al, 2005

30. Total Area of Echogenicity in the SN of Controls (49), Idiopathic RLS(39) and Secondary RLS (10) Subjects Control iRLS sRLS Godau et al, 2007

31. The Correlation between MRI–determined Iron in the Brain and Ultrasound- determined Iron in the Substantia Nigra Godau et al, 2008

32. General Findings of Transcranial Sonography in RLS  SN hypoechogenicity is not correlated with age of symptom onset (Godau 2008, Schmidauer 2005)  SN hypoechogenicity was more frequent in Idiopathic RLS (89%)then in secondary RLS (60%)  Red Nucleus showed Hyperechogenicity in RLS subjects (Godau 2008)  SN hypoechogenicity is not correlated with age of symptom onset (Godau 2008, Schmidauer 2005)  SN hypoechogenicity was more frequent in Idiopathic RLS (89%)then in secondary RLS (60%)  Red Nucleus showed Hyperechogenicity in RLS subjects (Godau 2008)

33. Summary  Overall RLS patients appears to have low CNS iron irrespective of the body iron stores.  Brain iron stores in RLS may be more independent of body iron stores and change more significantly over the circadian cycle  Early-onset vs late-onset RLS may differ, however, in the underlying pathology as regards iron.  Overall RLS patients appears to have low CNS iron irrespective of the body iron stores.  Brain iron stores in RLS may be more independent of body iron stores and change more significantly over the circadian cycle  Early-onset vs late-onset RLS may differ, however, in the underlying pathology as regards iron.

34. Summary  CSF: –Early AM sample; reliable, easy to perform and biggest effect size. Broadest iron assessment –Choroid vs tissue assessment  MRI: –R2’ GESFIDE most reliable for determination of tissue iron –Limited to tissue ferritin  Ultrasound: –Iron in tissue –Very tech–dependent  CSF: –Early AM sample; reliable, easy to perform and biggest effect size. Broadest iron assessment –Choroid vs tissue assessment  MRI: –R2’ GESFIDE most reliable for determination of tissue iron –Limited to tissue ferritin  Ultrasound: –Iron in tissue –Very tech–dependent