the prospective mri part of the study was approved by the local ethics committee ( ea 1/054/09 ) .
patient 1 is a 62-year old man who was admitted in 2008 because of subacute onset diplopia , dysphagia , and balance problems ( table ) .
neurologic examination revealed gaze - evoked nystagmus , diplopia , limb and gait ataxia , and moderate postural tremor of the upper extremities .
mri showed patchy t2 signal hyperintensities in the brainstem and cerebellar peduncles with punctate gadolinium enhancements ( figure 1 ) .
three lumbar punctures during the disease course revealed mild lymphocytic pleocytosis ( 37 , 17 , and 15 leukocytes per microliter ) but no oligoclonal bands .
there was no evidence of malignant cells and viral examinations of the csf for herpesviruses were negative .
search for onconeural antibodies , antinuclear antibodies , antineutrophil cytoplasmic antibodies , anti - ssa , anti - ssb , anticardiolipin , elevated angiotensin - converting enzyme in sera and csf , and tuberculosis tests were normal .
the patient recovered on high - dose steroid treatment , but relapsed after its withdrawal .
there was no evidence of lymphoma or vessel destruction . staining for herpes simplex virus 1 and 2 were normal .
the patient responded to steroid treatment , but then had a fatal brainstem hemorrhage . in 2014 , the history and initial brain biopsy specimens were re - evaluated and clippers syndrome was diagnosed .
demographics , clinical symptoms , treatment , and timing of mri or neuropathologic investigations of patients in this study ( a ) mri of patient 1 shows t2-weighted hyperintensities in brainstem and cerebellar peduncles with similar pattern of gadolinium enhancement .
arrows depict areas used for immunohistochemical stainings for cd45 , cd3 , cd4 , cd8 , and cd20 in brainstem ( b  f ) and cerebellum ( l  p ) with punctate gadolinium enhancements and parietal ( g  k ) and insular cortex ( q  u ) with normal mri appearance .
we found prominent cd4 + t - cell infiltration in the brainstem ( d ) and cerebellum ( n ) but also in insular cortex ( s ) and parietal cortex ( i ) .
the magnitude of infiltration shows a gradient with less infiltration with greater distance from the brainstem .
similarly , we found less infiltration of cd8 + t cells ( e , j , o , and t ) and only a very limited number of cd20 + b cells ( f , k , p , and u ) with a similar gradient in cell numbers and degree of infiltration into the tissue .
hematoxylin & eosin staining reveals inflammation also in cranial nerve roots from the brainstem ( v and w ) .
magnification 5 ( b and f ) ( v and w ) , 10 ( g  u ) .
scale bar : 200 m ( b  f ) , 100 m ( g  u ) , 200 m ( v , w ) . the clinical course , mri data , and follow - up of the 3 patients have been recently described elsewhere .
patient 2 is a 23-year - old man who developed subacute nystagmus and gait ataxia ( table ) .
he was treated with methylprednisolone and azathioprine , but had 6 relapses in 14 years with gradual worsening of cerebellar dysarthria and gait ataxia .
no systemic autoantibodies were found . at present , the patient is ambulatory ( modified rankin scale [ mrs ] 2 ) .
patient 3 is a 58-year - old woman who developed subacute gait ataxia , dysarthria , and paresthesia of the face , hands , and feet ( table ) .
brain biopsy indicated parenchymal and perivascular inflammatory infiltrates predominantly composed of cd3 + t cells .
no systemic autoantibodies were found . at present , the patient is severely dysarthric , wheelchair - bound , and has mild involuntary movements ( mrs 4 ) .
patient 4 is a 42-year - old man who developed subacute dysarthria , horizontal nystagmus , diplopia , jerky eye movements , ataxia , tetraspasticity , and paraparesis ( table ) .
brain biopsy indicated parenchymal and perivascular inflammatory infiltrates predominantly composed of cd3 + t cells .
mri showed multiple punctate gadolinium - enhancing lesions in the cerebellum , brainstem , and midbrain .
he was treated with methotrexate and azathioprine , and had only a single relapse during the follow - up period of 19 months ( mrs 2 ) .
patient 5 is a 48-year - old woman who developed subacute numbness in the left half of her body , double vision , gait instability , dysarthria , facial pain , numbness of the oral cavity , and fatigue over a period of 2 weeks in september 2013 ( table ) . during the weeks before the onset of these symptoms
the patient was admitted to another hospital in october 2013 , where a cranial 1.5 t mri demonstrated multiple small t2 hyperintense lesions with punctate and curvilinear contrast enhancement in the pons and cerebellum , and a bigger right cerebellar lesion . as this case
has not been published before , the brain mri findings of this patient are shown in figure e-1 at neurology.org/nn .
a spinal cord mri demonstrated a small longitudinal contrast - enhancing lesion in the cervical cord .
histologic analysis revealed cerebellitis with prominent cd4 + t - cellular infiltrates , compatible with clippers syndrome .
antinuclear antibodies , antibodies to extractable nuclear antigens , and antineutrophil cytoplasmic antibodies as well as antibodies to aquaporin-4 were negative .
she was initially treated with high - dose iv methylprednisolone ( 1 g / day for 3 days followed by 1 g / day for 5 days ) and subsequently with oral prednisolone ( starting dose 25 mg / day with a subsequent taper to 5 mg / day ) and azathioprine ( 3  50 mg ) , which was changed to methotrexate in may 2014 , when a cranial mri demonstrated constant to minimally progressive clippers lesions . at the time of the 7.0 t mri examination , she was treated with prednisolone ( 5 mg daily ) and methotrexate ( 15 mg weekly ) , and had residual facial paresthesiae but no clinical signs of new disease activity .
patient 6 is a 73-year - old woman who was diagnosed with clippers in october 2010 and whose clinical and paraclinical details were previously reported in detail .
the patient had mildly elevated antinuclear antibodies ( 1:320 ) and double - stranded dna antibodies ( immunoglobulin g ) .
at the time of the 7.0 t mri examination , she was treated with methylprednisolone ( 4 mg daily ) and methotrexate ( 15 mg weekly ) and was in clinical remission .
three - micrometer sections from formalin - fixed , paraffin - embedded tissue blocks were stained with hematoxylin & eosin ( h&e ) and for cd45 ( clone : 2b11&pd7/26 ventana - lca , ventana medical systems , tucson , az ) , cd3 ( clone : 2gv6 , ventana medical systems ) , cd4 ( sp35 , ventana medical systems ) , cd8 ( clone : c8/144b , dako , glostrup , denmark ) , cd20 ( clone : cyl26 , ventana medical systems ) , and neurofilament heavy - chain ( nf smi31 , affinity , exeter , uk ) . selected sections from brainstem and insular
adjacent formalin - fixed tissue section from brainstem tissue ( autopsy ) as well as sections from 3 additional brainstem biopsies were similarly stained for light - chain restriction ( kappa - smi and lambda - smi , smlg reaction , dako ) , the zinc - finger transcription factor , b - cell lymphoma 6 protein ( bcl-6 , clone : ln22 novocastra , leica biosystems , newcastle , uk ) , the large b - cell lymphoma marker , multiple myeloma oncogene 1 ( mum-1 , irf4 , dako ) , and the precursor b lymphoma marker , cd10 ( clone : 56c6-calla , novocastra , leica biosystems ) .
all immunohistochemical stainings were performed using the benchmark ultra ihc staining system ( ventana medical systems ) . for detection ,
digital images were obtained at a magnification of 5 or 10 ( brainstem ) or 10 ( cerebellum , parietal cortex , insular cortex / basal ganglia , nerve root , and biopsies 13 ) using a leica microscope ( leica 4000b led , leica microsystems , wetzlar , germany ) equipped with a leica digital camera ( leica dfc420 , leica microsystems ) .
ultra - high - field mri were acquired using a 7.0 t whole body mri scanner ( magnetom , siemens , erlangen , germany ) applying a 24-channel receive head coil ( nova medical , wilmington , ma ) equipped with a birdcage volume coil used for transmission .
the imaging protocol included 2d t2 * -weighted ( t2*w ) fast low angle shot ( echo time [ te ] = 25.0 ms , repetition time [ tr ] = 1,820 ms , spatial resolution = 0.2  0.2  2 mm , supratentorial coverage ) , pregadolinium and postgadolinium 3d t1-weighted magnetization - prepared rapid gradient echo ( t1w mprage ) ( te = 2.98 ms , tr = 2,300 ms , inversion time = 900 ms , spatial resolution = 1.0  1.0  1.0 mm , whole brain coverage ) , and 3d flow - compensated gradient echo  based susceptibility - weighted imaging ( swi ) ( te = 14 ms , tr = 25 ms , flip angle = 12 , spatial resolution = 0.5  0.5  1.0 mm , supratentorial and infratentorial coverage ) . for infratentorial imaging , postgadolinium volumetric interpolated brain examination ( vibe ) sequence was used ( te = 3.3 ms , tr = 19.7 ms , spatial resolution = 0.5  0.5  1.0 mm ) . to identify vascular structures within contrast - enhancing clippers lesions , we calculated the sum of ( 1 ) coregistered t1w mprage with supratentorial t2*w images , and ( 2 ) coregistered vibe with infratentorial swi images using the image calculator function of the mipav software package ( version 7.0.1 , bethesda , md ) .
the prospective mri part of the study was approved by the local ethics committee ( ea 1/054/09 ) .
patient 1 is a 62-year old man who was admitted in 2008 because of subacute onset diplopia , dysphagia , and balance problems ( table ) .
neurologic examination revealed gaze - evoked nystagmus , diplopia , limb and gait ataxia , and moderate postural tremor of the upper extremities .
mri showed patchy t2 signal hyperintensities in the brainstem and cerebellar peduncles with punctate gadolinium enhancements ( figure 1 ) .
three lumbar punctures during the disease course revealed mild lymphocytic pleocytosis ( 37 , 17 , and 15 leukocytes per microliter ) but no oligoclonal bands .
there was no evidence of malignant cells and viral examinations of the csf for herpesviruses were negative .
search for onconeural antibodies , antinuclear antibodies , antineutrophil cytoplasmic antibodies , anti - ssa , anti - ssb , anticardiolipin , elevated angiotensin - converting enzyme in sera and csf , and tuberculosis tests were normal .
the patient recovered on high - dose steroid treatment , but relapsed after its withdrawal .
there was no evidence of lymphoma or vessel destruction . staining for herpes simplex virus 1 and
the patient responded to steroid treatment , but then had a fatal brainstem hemorrhage . in 2014 ,
the history and initial brain biopsy specimens were re - evaluated and clippers syndrome was diagnosed .
demographics , clinical symptoms , treatment , and timing of mri or neuropathologic investigations of patients in this study ( a ) mri of patient 1 shows t2-weighted hyperintensities in brainstem and cerebellar peduncles with similar pattern of gadolinium enhancement .
arrows depict areas used for immunohistochemical stainings for cd45 , cd3 , cd4 , cd8 , and cd20 in brainstem ( b  f ) and cerebellum ( l  p ) with punctate gadolinium enhancements and parietal ( g  k ) and insular cortex ( q  u ) with normal mri appearance .
we found prominent cd4 + t - cell infiltration in the brainstem ( d ) and cerebellum ( n ) but also in insular cortex ( s ) and parietal cortex ( i ) .
the magnitude of infiltration shows a gradient with less infiltration with greater distance from the brainstem .
similarly , we found less infiltration of cd8 + t cells ( e , j , o , and t ) and only a very limited number of cd20 + b cells ( f , k , p , and u ) with a similar gradient in cell numbers and degree of infiltration into the tissue .
hematoxylin & eosin staining reveals inflammation also in cranial nerve roots from the brainstem ( v and w ) .
magnification 5 ( b and f ) ( v and w ) , 10 ( g  u ) .
scale bar : 200 m ( b  f ) , 100 m ( g  u ) , 200 m ( v , w ) .
the clinical course , mri data , and follow - up of the 3 patients have been recently described elsewhere .
patient 2 is a 23-year - old man who developed subacute nystagmus and gait ataxia ( table ) .
he was treated with methylprednisolone and azathioprine , but had 6 relapses in 14 years with gradual worsening of cerebellar dysarthria and gait ataxia .
no systemic autoantibodies were found . at present , the patient is ambulatory ( modified rankin scale [ mrs ] 2 ) .
patient 3 is a 58-year - old woman who developed subacute gait ataxia , dysarthria , and paresthesia of the face , hands , and feet ( table ) .
brain biopsy indicated parenchymal and perivascular inflammatory infiltrates predominantly composed of cd3 + t cells .
no systemic autoantibodies were found . at present , the patient is severely dysarthric , wheelchair - bound , and has mild involuntary movements ( mrs 4 ) .
patient 4 is a 42-year - old man who developed subacute dysarthria , horizontal nystagmus , diplopia , jerky eye movements , ataxia , tetraspasticity , and paraparesis ( table ) .
brain biopsy indicated parenchymal and perivascular inflammatory infiltrates predominantly composed of cd3 + t cells .
mri showed multiple punctate gadolinium - enhancing lesions in the cerebellum , brainstem , and midbrain .
he was treated with methotrexate and azathioprine , and had only a single relapse during the follow - up period of 19 months ( mrs 2 ) .
patient 5 is a 48-year - old woman who developed subacute numbness in the left half of her body , double vision , gait instability , dysarthria , facial pain , numbness of the oral cavity , and fatigue over a period of 2 weeks in september 2013 ( table ) . during the weeks before the onset of these symptoms
the patient was admitted to another hospital in october 2013 , where a cranial 1.5 t mri demonstrated multiple small t2 hyperintense lesions with punctate and curvilinear contrast enhancement in the pons and cerebellum , and a bigger right cerebellar lesion . as this case
has not been published before , the brain mri findings of this patient are shown in figure e-1 at neurology.org/nn .
a spinal cord mri demonstrated a small longitudinal contrast - enhancing lesion in the cervical cord .
histologic analysis revealed cerebellitis with prominent cd4 + t - cellular infiltrates , compatible with clippers syndrome .
antinuclear antibodies , antibodies to extractable nuclear antigens , and antineutrophil cytoplasmic antibodies as well as antibodies to aquaporin-4 were negative .
she was initially treated with high - dose iv methylprednisolone ( 1 g / day for 3 days followed by 1 g / day for 5 days ) and subsequently with oral prednisolone ( starting dose 25 mg / day with a subsequent taper to 5 mg / day ) and azathioprine ( 3  50 mg ) , which was changed to methotrexate in may 2014 , when a cranial mri demonstrated constant to minimally progressive clippers lesions . at the time of the 7.0 t mri examination , she was treated with prednisolone ( 5 mg daily ) and methotrexate ( 15 mg weekly ) , and
patient 6 is a 73-year - old woman who was diagnosed with clippers in october 2010 and whose clinical and paraclinical details were previously reported in detail .
the patient had mildly elevated antinuclear antibodies ( 1:320 ) and double - stranded dna antibodies ( immunoglobulin g ) .
antibodies against aquaporin-4 and extractable nuclear antigens were negative . at the time of the 7.0 t mri examination , she was treated with methylprednisolone ( 4 mg daily ) and methotrexate ( 15 mg weekly ) and was in clinical remission .
three - micrometer sections from formalin - fixed , paraffin - embedded tissue blocks were stained with hematoxylin & eosin ( h&e ) and for cd45 ( clone : 2b11&pd7/26 ventana - lca , ventana medical systems , tucson , az ) , cd3 ( clone : 2gv6 , ventana medical systems ) , cd4 ( sp35 , ventana medical systems ) , cd8 ( clone : c8/144b , dako , glostrup , denmark ) , cd20 ( clone : cyl26 , ventana medical systems ) , and neurofilament heavy - chain ( nf smi31 , affinity , exeter , uk ) . selected sections from brainstem and insular
adjacent formalin - fixed tissue section from brainstem tissue ( autopsy ) as well as sections from 3 additional brainstem biopsies were similarly stained for light - chain restriction ( kappa - smi and lambda - smi , smlg reaction , dako ) , the zinc - finger transcription factor , b - cell lymphoma 6 protein ( bcl-6 , clone : ln22 novocastra , leica biosystems , newcastle , uk ) , the large b - cell lymphoma marker , multiple myeloma oncogene 1 ( mum-1 , irf4 , dako ) , and the precursor b lymphoma marker , cd10 ( clone : 56c6-calla , novocastra , leica biosystems ) .
all immunohistochemical stainings were performed using the benchmark ultra ihc staining system ( ventana medical systems ) . for detection ,
digital images were obtained at a magnification of 5 or 10 ( brainstem ) or 10 ( cerebellum , parietal cortex , insular cortex / basal ganglia , nerve root , and biopsies 13 ) using a leica microscope ( leica 4000b led , leica microsystems , wetzlar , germany ) equipped with a leica digital camera ( leica dfc420 , leica microsystems ) .
ultra - high - field mri were acquired using a 7.0 t whole body mri scanner ( magnetom , siemens , erlangen , germany ) applying a 24-channel receive head coil ( nova medical , wilmington , ma ) equipped with a birdcage volume coil used for transmission .
the imaging protocol included 2d t2 * -weighted ( t2*w ) fast low angle shot ( echo time [ te ] = 25.0 ms , repetition time [ tr ] = 1,820 ms , spatial resolution = 0.2  0.2  2 mm , supratentorial coverage ) , pregadolinium and postgadolinium 3d t1-weighted magnetization - prepared rapid gradient echo ( t1w mprage ) ( te = 2.98 ms , tr = 2,300 ms , inversion time = 900 ms , spatial resolution = 1.0  1.0  1.0 mm , whole brain coverage ) , and 3d flow - compensated gradient echo  based susceptibility - weighted imaging ( swi ) ( te = 14 ms , tr = 25 ms , flip angle = 12 , spatial resolution = 0.5  0.5  1.0 mm , supratentorial and infratentorial coverage ) . for infratentorial imaging , postgadolinium volumetric interpolated brain examination ( vibe ) sequence
was used ( te = 3.3 ms , tr = 19.7 ms , spatial resolution = 0.5  0.5  1.0 mm ) . to identify vascular structures within contrast - enhancing clippers lesions , we calculated the sum of ( 1 ) coregistered t1w mprage with supratentorial t2*w images , and ( 2 ) coregistered vibe with infratentorial swi images using the image calculator function of the mipav software package ( version 7.0.1 , bethesda , md ) .
first , we investigated inflammation in different brain regions corresponding to gadolinium - enhancing and normal - appearing brain areas on 3.0 t mri ( autopsy tissue of patient 1 ) : brainstem and cerebellum were compared to parietal and insular cortex ( figure 1 ) .
perivascular accumulation of cd45 + lymphocytes ( figure 1 , b , g , l , and q ) with predominance of cd3 + t cells ( figure 1 , c , h , m and r ) and cd4 + t cells were detected in brainstem and cerebellum as expected ( figure 1 , d and n ) .
cd4 + t cells also infiltrated the parenchyma , which was most pronounced in the brainstem ( figure 1d ) .
accumulation and infiltration by cd8 + t cells were less pronounced ( figure 1 , e and q ) , and only a few cd20 + b cells were observed in close proximity to the vessel wall ( figure 1 , f and p ) . however , insular and parietal cortex with normal appearance on 3.0 t mri revealed a similar , albeit less prominent perivascular accumulation of cd4 + t cells ( figure 1 , i and s ) , fewer cd8 + t cells ( figure 1 , j and t ) , and a very limited number of cd20 + b cells ( figure 1 , k and u ) .
cd4 + t cells also infiltrated the parenchyma the insular cortex ( figure 1s ) .
perivascular inflammation was also found in the roots of cranial nerves ( figure 1 , v and w ) .
these data suggested that brain areas with normal appearance on 3.0 t mri may be affected by inflammation in clippers syndrome .
next , brainstem samples ( patients 14 ) were analyzed for the possibility of lymphoma or prelymphoma .
we found no evidence for monoclonal cell expansion using staining for light chain restriction ( figure 2 , a  b , f 
l , and p  q ) and lymphoma markers cd10 , bcl-6 , and mum-1 ( figure 2 , c  e , h  j , m  o , and r 
follow - up of the 3 biopsy cases ( patients 24 ; mean of 75 months ) did not indicate clinical or paraclinical evidence of lymphoma either .
immunohistochemical stainings for light chain restriction ( lambda - smi and kappa - smi : a , b , f , g , k , l , p , and q ) , cd10 ( c , h , m , and r ) , bcl-6 ( d , i , n , and s ) , and mum-1 ( e , j , o , and t ) in samples from an autopsy ( a  e ) and biopsy specimens of 3 patients ( f  j , k 
we found no evidence for light chain restriction or significant staining for lymphoma markers cd10 , bcl-6 , or mum-1 suggesting a prelymphoma or lymphoma state .
scale bar : 200 m ( a  e ) , 100 m ( f  t ) .
since pathologic examination indicated perivascular inflammation in brain areas with normal appearance on 3.0 t mri , albeit less pronounced than in brainstem and cerebellum , we next examined 2 patients with clippers by 7.0 t mri during clinical remission . contrast - enhancing lesions ( n = 20 ) were only depicted in patient 5 . of those , 2 lesions were found in a supratentorial localization affecting ( n = 1 ) or bordering ( n = 1 ) the thalamus ( figure 3d ) .
supratentorial t2 * -weighted ( a , b ) and postgadolinium t1-weighted images ( d ) of patient 5 obtained at 7.0 t are displayed .
postgadolinium t1-weighted imaging depicts a small contrast - enhancing lesion ( black arrow , d ) that is only marginally delineated on corresponding t2 * -weighted images ( zoom , b ) despite using a very high spatial resolution of 0.08 mm .
fusion ( c ) of coregistered postgadolinium t1-weighted ( d ) and t2 * -weighted ( b ) identifies a small brain vessel within the center of the chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids ( clippers ) lesion ( white arrow , c ) .
* -weighted fast low angle shot at 7.0 t , echo time ( te ) = 25.0 ms , repetition time ( tr ) = 1,820 ms , spatial resolution = 0.2  0.2  2 mm ; t1-gad = postgadolinium 3d t1-weighted magnetization - prepared rapid gradient echo at 7.0 t , te = 2.98 ms , tr = 2,300 ms , inversion time = 900 ms , spatial resolution = 1.0  1.0  1.0 mm . to visualize the perivascular distribution of lesions
, we fused contrast - enhanced t1w and supratentorial t2*w images with a volume resolution of 0.08 mm to display small t2*w hypointense venous structures within gadolinium - enhancing hyperintense clippers lesions ( figure 3 ) .
indeed , a distinct venous vessel could be seen within the center of the thalamic clippers lesion in vivo ( figure 3c ) .
due to technical limitations , a different approach with a lower spatial resolution of 0.25 mm was used for infratentorial areas .
even with this approach , a very small venous vessel could be depicted within 8 of 10 contrast - enhancing clippers lesions on fused postgadolinium vibe and swi ( data not shown ) .
owing to substantially increased signal - to - noise ratio , 7.0 t t1-weighted magnetization - prepared rapid gradient echo delineates numerous t1-weighted hypointense lesions within the pons and cerebellum ( arrowheads , e 
in contrast , corresponding areas appear normal ( c , d ) or only marginally hypointense on t1-weighted images at 3.0 t ( circle , a , b ) .
t1-weighted hypointensity within non - contrast - enhancing chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids lesions may indicate postinflammatory structural brain damage including axonal loss on top of demyelination .
immunohistochemical stainings of autopsy case for neurofilament ( nf ) revealed massive axonal injury in the inflammatory lesion of the brainstem ( h ; cd3 red , nf brown ) compared to insular cortex with limited inflammation ( j ) .
the presence of phosphorylated nf in the perinuclear cytoplasm of neurons suggests retrograde neuronal degeneration due to axonal injury in the lesions ( h ) .
similarly , severe loss of perivascular myelin was found in areas of severe inflammation ( i ) , compared to areas with limited inflammation ( k ) , with luxol fast blue and hematoxylin & eosin .
7.0 t = 3d t1-weighted magnetization - prepared rapid gradient echo at 7.0 t , echo time = 2.98 ms , repetition time = 2,300 ms , inversion time = 900 ms , spatial resolution = 1.0  1.0  1.0 mm ; 3.0 t = standard t1-weighted axial images ( b  d ) and t1-weighted magnetization - prepared rapid gradient echo at 3.0 t ( a ) .
scale bar = 100 m . in addition , 7.0 t t1w mprage depicted numerous t1w hypointense lesions within the pons and cerebellum ( figure 4 , e 
g ) in patient 5 , which were not visible on conventional 3.0 t mri ( figure 4 , a 
d ) , indicating severe tissue destruction within postinflammatory clippers lesions ( figure 4 ) .
multiple pontine postinflammatory t1w hypointense lesions were also found in patient 6 by using t1w imaging at 7.0 t ( data not shown ) .
corresponding to the 7.0 t mri findings , neurofilament and myelin staining revealed axonal injury and myelin loss in brainstem areas with severe inflammation of the autopsy case ( figure 4 , h and i ) .
preservation of axons and myelin was seen in areas with limited inflammation ( figure 4 , j and k ) .
first , we investigated inflammation in different brain regions corresponding to gadolinium - enhancing and normal - appearing brain areas on 3.0 t mri ( autopsy tissue of patient 1 ) : brainstem and cerebellum were compared to parietal and insular cortex ( figure 1 ) .
perivascular accumulation of cd45 + lymphocytes ( figure 1 , b , g , l , and q ) with predominance of cd3 + t cells ( figure 1 , c , h , m and r ) and cd4 + t cells were detected in brainstem and cerebellum as expected ( figure 1 , d and n ) .
cd4 + t cells also infiltrated the parenchyma , which was most pronounced in the brainstem ( figure 1d ) .
accumulation and infiltration by cd8 + t cells were less pronounced ( figure 1 , e and q ) , and only a few cd20 + b cells were observed in close proximity to the vessel wall ( figure 1 , f and p ) . however , insular and parietal cortex with normal appearance on 3.0 t mri revealed a similar , albeit less prominent perivascular accumulation of cd4 + t cells ( figure 1 , i and s ) , fewer cd8 + t cells ( figure 1 , j and t ) , and a very limited number of cd20 + b cells ( figure 1 , k and u ) .
cd4 + t cells also infiltrated the parenchyma the insular cortex ( figure 1s ) .
perivascular inflammation was also found in the roots of cranial nerves ( figure 1 , v and w ) .
these data suggested that brain areas with normal appearance on 3.0 t mri may be affected by inflammation in clippers syndrome .
next , brainstem samples ( patients 14 ) were analyzed for the possibility of lymphoma or prelymphoma .
we found no evidence for monoclonal cell expansion using staining for light chain restriction ( figure 2 , a  b , f 
l , and p  q ) and lymphoma markers cd10 , bcl-6 , and mum-1 ( figure 2 , c  e , h  j , m 
follow - up of the 3 biopsy cases ( patients 24 ; mean of 75 months ) did not indicate clinical or paraclinical evidence of lymphoma either .
immunohistochemical stainings for light chain restriction ( lambda - smi and kappa - smi : a , b , f , g , k , l , p , and q ) , cd10 ( c , h , m , and r ) , bcl-6 ( d , i , n , and s ) , and mum-1 ( e , j , o , and t ) in samples from an autopsy ( a  e ) and biopsy specimens of 3 patients ( f  j , k 
we found no evidence for light chain restriction or significant staining for lymphoma markers cd10 , bcl-6 , or mum-1 suggesting a prelymphoma or lymphoma state .
scale bar : 200 m ( a  e ) , 100 m ( f  t ) .
since pathologic examination indicated perivascular inflammation in brain areas with normal appearance on 3.0 t mri , albeit less pronounced than in brainstem and cerebellum , we next examined 2 patients with clippers by 7.0 t mri during clinical remission . contrast - enhancing lesions ( n = 20 ) were only depicted in patient 5 . of those , 2 lesions were found in a supratentorial localization affecting ( n = 1 ) or bordering ( n = 1 ) the thalamus ( figure 3d ) .
supratentorial t2 * -weighted ( a , b ) and postgadolinium t1-weighted images ( d ) of patient 5 obtained at 7.0 t are displayed .
postgadolinium t1-weighted imaging depicts a small contrast - enhancing lesion ( black arrow , d ) that is only marginally delineated on corresponding t2 * -weighted images ( zoom , b ) despite using a very high spatial resolution of 0.08 mm .
fusion ( c ) of coregistered postgadolinium t1-weighted ( d ) and t2 * -weighted ( b ) identifies a small brain vessel within the center of the chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids ( clippers ) lesion ( white arrow , c ) .
t2*w = 2d t2 * -weighted fast low angle shot at 7.0 t , echo time ( te ) = 25.0 ms , repetition time ( tr ) = 1,820 ms , spatial resolution = 0.2  0.2  2 mm ; t1-gad = postgadolinium 3d t1-weighted magnetization - prepared rapid gradient echo at 7.0 t , te = 2.98 ms , tr = 2,300 ms , inversion time = 900 ms , spatial resolution = 1.0  1.0  1.0 mm . to visualize the perivascular distribution of lesions
, we fused contrast - enhanced t1w and supratentorial t2*w images with a volume resolution of 0.08 mm to display small t2*w hypointense venous structures within gadolinium - enhancing hyperintense clippers lesions ( figure 3 ) .
indeed , a distinct venous vessel could be seen within the center of the thalamic clippers lesion in vivo ( figure 3c ) . due to technical limitations , a different approach with a lower spatial resolution of 0.25 mm
even with this approach , a very small venous vessel could be depicted within 8 of 10 contrast - enhancing clippers lesions on fused postgadolinium vibe and swi ( data not shown ) .
t1-weighted images of patient 5 at 3.0 t and 7.0 t are shown . owing to substantially increased signal - to - noise ratio , 7.0 t t1-weighted
magnetization - prepared rapid gradient echo delineates numerous t1-weighted hypointense lesions within the pons and cerebellum ( arrowheads , e 
in contrast , corresponding areas appear normal ( c , d ) or only marginally hypointense on t1-weighted images at 3.0 t ( circle , a , b ) .
t1-weighted hypointensity within non - contrast - enhancing chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids lesions may indicate postinflammatory structural brain damage including axonal loss on top of demyelination .
immunohistochemical stainings of autopsy case for neurofilament ( nf ) revealed massive axonal injury in the inflammatory lesion of the brainstem ( h ; cd3 red , nf brown ) compared to insular cortex with limited inflammation ( j ) .
the presence of phosphorylated nf in the perinuclear cytoplasm of neurons suggests retrograde neuronal degeneration due to axonal injury in the lesions ( h ) .
similarly , severe loss of perivascular myelin was found in areas of severe inflammation ( i ) , compared to areas with limited inflammation ( k ) , with luxol fast blue and hematoxylin & eosin .
7.0 t = 3d t1-weighted magnetization - prepared rapid gradient echo at 7.0 t , echo time = 2.98 ms , repetition time = 2,300 ms , inversion time = 900 ms , spatial resolution = 1.0  1.0  1.0 mm ; 3.0 t = standard t1-weighted axial images ( b  d ) and t1-weighted magnetization - prepared rapid gradient echo at 3.0 t ( a ) .
in addition , 7.0 t t1w mprage depicted numerous t1w hypointense lesions within the pons and cerebellum ( figure 4 , e 
g ) in patient 5 , which were not visible on conventional 3.0 t mri ( figure 4 , a 
d ) , indicating severe tissue destruction within postinflammatory clippers lesions ( figure 4 ) .
multiple pontine postinflammatory t1w hypointense lesions were also found in patient 6 by using t1w imaging at 7.0 t ( data not shown ) .
corresponding to the 7.0 t mri findings , neurofilament and myelin staining revealed axonal injury and myelin loss in brainstem areas with severe inflammation of the autopsy case ( figure 4 , h and i ) .
preservation of axons and myelin was seen in areas with limited inflammation ( figure 4 , j and k ) .
in this article , we describe neuropathologic as well as high - field / ultra - high - field mri features of 6 patients with clippers syndrome .
the diagnosis of clippers syndrome was supported by ( 1 ) postcontrast t1-weighted mri with typical punctate , nodular , or curvilinear and occasionally more confluent gadolinium - enhancing lesions , ( 2 ) perivascular inflammation in the brainstem dominated by t cells , ( 3 ) clinical course with several steroid - responsive relapses , and ( 4 ) absence of other diseases .
in addition to punctate and curvilinear lesions , case 5 had a larger confluent lesion in the cerebellum .
similar lesions were previously described in patients with clippers and a biopsy of the cerebellar lesion of case 5 demonstrated histopathologic features of clippers .
we found widespread perivascular inflammation in brain areas with normal appearance on 3.0 t mri in an autopsy case of clippers syndrome .
the inflammatory pattern of these regions was similar to the original and later neuropathologic descriptions of the disease characterized by pronounced cd4 + t - cell infiltration of brain tissue and less predominant angiocentric cd8 + t - cell and cd20 + b - cell inflammation .
although t2w and gadolinium - enhancing lesions were found only in the pons , midbrain , and cerebellar regions , inflammation could be seen as distant as the parietal cortex and even involved the cranial nerve roots .
the degree of inflammation , however , showed a gradient of less inflammation with greater distance from the primarily affected areas ( brainstem > cerebellum > insular cortex > parietal cortex ) , suggesting that the inflammation seen on conventional 1.5t3.0 t mri only depicts the most severely affected brain regions .
consistent with these neuropathologic findings , cognitive impairment such as dysexecutive syndromes , language disturbance , frontal release signs and cortical atrophy were described in a case series of clippers syndrome , suggesting that cortical involvement may be more prominent than initially thought .
since our pathologic examinations revealed widespread inflammatory changes in brain tissue appearing normal on conventional 3.0 t mri , we performed ultra - high - field 7.0 t mri in 2 additional clippers cases to investigate such possibilities in vivo .
indeed , these efforts revealed contrast - enhancing lesions in supratentorial locations , supporting widespread inflammation in brain areas , in contrast to the only other autopsy report published recently .
the number of such contrast - enhancing lesions was , however , limited , which is likely due to the immunosuppressive treatment : mri changes in clippers respond rapidly to immunosuppression . remarkably , when combining t1w and supratentorial t2*w images with a resolution as good as 0.08 mm , we could show a vascular structure within most contrast - enhancing lesions .
the 7.0 t mri thus enabled visualization of perivascular inflammation , the neuropathologic hallmark of clippers , in vivo .
the hypointense appearance of these intralesional vascular structures on t2*w and swi images indicates the presence of a paramagnetic material .
thus , these intralesional vascular structures are most likely small veins filled with paramagnetic deoxyhemoglobin .
a central intralesional vein was previously reported to be a characteristic finding in ms lesions . on the contrary ,
a central vein is only rarely depictable in brain lesions of other origin such as neuromyelitis optica , cerebral small vessel disease , and susac syndrome , which is considered a microangiopathy of the brain , cochlea , and retina .
evidence for the presence of mri - detectable intralesional veins in other inflammatory vasculopathies such as wegner granulomatosis , behet syndrome , primary cns vasculitis , and vasculitis in systemic lupus erythematosus is , however , limited . of note , highly resolving imaging techniques
were needed to visualize the intralesional venous vessel in clippers lesions , which thus appear much smaller in diameter than central veins commonly visualized in ms lesions on 7.0 t t2*w images . however , as no systematic analysis of vessel diameters in clippers vs ms could be performed owing to the low sample size , future comparative studies will have to investigate whether this feature is consistently detectable in other clippers cases and distinct from ms .
in addition , we utilized the higher sensitivity of 7.0 t t1w mprage vs t1w imaging at 1.5 t and 3.0 t to detect brain lesions and to investigate brain areas that appeared normal at lower field strengths .
although 1.5t3.0 t mri and 7.0 t mri were done at different time points , therefore direct comparison was not possible , patients had no signs of clinical activity at the time of the mri examinations .
ultra - high - field mri revealed numerous t1w hypointense lesions within the pons and cerebellum in one of the patients , indicating postinflammatory brain tissue destruction , which were not visible on conventional 3.0 t mri .
indeed , axonal injury with evidence of axonal spheroids and torpedoes was found in our autopsy case , similar to previous results .
however , axonal injury and myelin loss was observed only in areas with severe inflammation , which may explain the restricted localization of t1w hypointense lesions on 7.0 t mri . since emerging cases of clippers with subsequent diagnosis of lymphoma
have been recently described , we investigated this possibility in the autopsy case and available biopsy samples from 3 other danish cases .
we found no evidence for prelymphoma or lymphoma when using immunohistochemical staining for light chain restriction or lymphoma markers cd10 , bcl-6 , and mum-1 .
since the analyses were done on the initial diagnostic biopsies , this might not exclude per se that the patients progressed into a lymphoma .
however , 3 of the patients have been already followed for a mean of more than 6 years without any evidence of lymphoma . altogether , our present results strengthen the concept that clippers is a distinct disease entity that can be discerned from alternative diagnoses such as lymphoma or vasculitis
. we found perivascular inflammation predominated by cd4 + t cells even in areas with normal appearance on 3.0 t mri and extending to the cranial nerve roots , suggesting widespread inflammation in clippers syndrome , though most prominent in the primarily affected brainstem .
we found no evidence of lymphoma in our 4 investigated patients . when applying ultra - high - field mri at 7.0 t , supratentorial lesions
could be confirmed , and most of the contrast - enhancing lesions contained a vessel structure indicating the pathologic hallmark of clippers syndrome in vivo .
the 7.0 t mri also indicated prominent t1w hypointensities suggesting tissue injury , which could not be seen on 3.0 t mri but corresponded to axonal injury and loss of myelin in the autopsy specimen .
one limitation of the study might be the application of immunosuppressive treatment at the time of biopsy or mri examination ( table 1 ) .
morten blaabjerg : design and conceptualization , clinical data , analysis and interpretation of the data , drafting and revising the manuscript .
klemens ruprecht : mri design and conceptualization , clinical data , analysis and interpretation of the data , drafting and revising the manuscript .
bjrg morell kerrn - jespersen : clinical data , interpretation of the data , revising the manuscript .
bjarne winther kristensen : pathological analysis and interpretation of the data , revising the manuscript .
friedemann paul : mri design and conceptualization , analysis and interpretation of the data , drafting and revising the manuscript .
zsolt illes : design and conceptualization , analysis and interpretation of the data , drafting and revising the manuscript .
lundbeckfonden and scleroseforeningen ( denmark ) to z.i . ; deutsche forschungsgemeinschaft ( exc 257 ) and the german ministry for education and research ( bmbf competence network multiple sclerosis ) to f.p . ;
german ministry for education and research ( bmbf competence network multiple sclerosis ) to k.r .
k. ruprecht served on the scientific advisory board for sanofi - aventis / genzyme , novartis , and roche ; received travel funding and/or speaker honoraria from bayer healthcare , biogen idec , merck serono , sanofi - aventis / genzyme , teva pharmaceuticals , novartis , and guthy jackson charitable foundation ; is an academic editor for plos one ; receives publishing royalties from elsevier ; and received research support from novartis and the german ministry of education and research .
d. kondziella received speaker honoraria and/or travel funding from bristol meyer squibb and ucb and has consulted for pfizer sweden . t. niendorf received travel funding and/or speaker funding from siemens healthcare , is founder and ceo of mri tools gmbh , and received research support from siemens healthcare and helmholtz alliance .
lassmann received travel funding and speaker honoraria from biogen idec , novartis , and teva ; is on the editorial board for several journals in the fields of neurology and neuroscience ; has consulted for biogen idec and amgen ; and received research support from austrian science fund , european union .
f. paul is on the steering committees for novartis and medimmune ; received speaker honoraria and travel funding from bayer , novartis , biogen idec , teva , sanofi - aventis / genzyme , and merck serono ; is an academic editor for plos one ; is an associate editor for neurology : neuroimmunology & neuroinflammation ; has consulted for sanofigenzyme , biogenidec , and medimmune ; and received research support from bayer , novartis , biogen idec , teva , sanofi - aventis / genzyme , merck serono , german research council , werth stiftung of the city of cologne , german ministry of education and research , arthur arnstein stiftung berlin , eu fp7 framework program , guthy jackson charitable foundation , and national multiple sclerosis society of the usa .
z. illes served on the scientific advisory board for biogen idec , novartis , sanofi - aventis / genzyme , and teva pharmaceuticals ; received travel funding and/or speaker honoraria from biogen idec , novartis , sanofi - aventis / genzyme , teva pharmaceuticals , bayer healthcare , and merck serono ; is on the editorial board for clinical and experimental neuroimmunology ; holds a patent for fumaric acide derivates for medical use ; and received research support from biogen idec , region of southern denmark , odense university hospital , scleroseforeningen , lundbeckfonden , direktor enjar jonasson , kaldet jonsen , og hustru 's mindelegat .
 objective : to examine if there is widespread inflammation in the brain of patients with chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids ( clippers ) syndrome by using histology and ultra - high - field mri at 7.0t.methods:we performed a detailed neuropathologic examination in 4 cases , including 1 autopsy case , and studied 2 additional patients by mri at 7.0 t to examine ( 1 ) extension of inflammation to areas appearing normal on 3.0 t mri , ( 2 ) potential advantages of 7.0 t mri compared to 3.0 t mri in reflecting widespread inflammation , perivascular pathology , and axonal damage , and ( 3 ) the possibility of lymphoma.results:in the autopsy case , perivascular inflammation dominated by cd4 + t cells was not only detected in the brainstem and cerebellum but also in brain areas with normal appearance on 3.0 t mri , including supratentorial regions and cranial nerve roots . 
 there was no evidence of lymphoma in any of the 4 patients . 
 the 7.0 t mri in clinical remission also revealed supratentorial lesions and perivascular pathology in vivo with contrast - enhancing lesions centered around a small venous vessel . 
 ultra - high - field mri at 7.0 t disclosed prominent t1 hypointensities in the brainstem , which were not seen on 3.0 t mri . this corresponded to neuropathologic detection of axonal injury in the autopsy case.conclusion:our findings suggest more widespread perivascular inflammation and postinflammatory axonal injury in patients with clippers . 
METHODS
Standard approvals, registrations, and patients.
Description of autopsy case (patient 1).
Description of the 3 cases with brainstem biopsy (patients 24).
Patients examined by 7T MRI (patients 5 and 6).
Neuropathologic examination.
Ultra-high-field MRI data acquisition and postprocessing.
RESULTS
Perivascular inflammation in CLIPPERS extends to areas with normal appearance on 3.0T MRI: Analysis of the autopsy case.
No evidence of lymphoma and prelymphoma state: Analysis of 4 cases.
Ultra-high-field MRI reveals perivascular lesions outside the brainstem/cerebellum and tissue damage.
DISCUSSION
Supplementary Material
AUTHOR CONTRIBUTIONS
STUDY FUNDING
DISCLOSURE