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Is rat hippocampus section immunostaining an indicator for immunotherapy in cryptogenic adult new-onset refractory status epilepticus (NORSE)?
Seizure: European Journal of Epilepsy 76 (2020) 131–136
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Seizure: European Journal of Epilepsy journal homepage: www.elsevier.com/locate/seizure
Is rat hippocampus section immunostaining an indicator for immunotherapy in cryptogenic adult new-onset refractory status epilepticus (NORSE)?
T
Dongmei Wang1, Yue Pan1, Kaibin Huang, Zhenzhou Lin, Zuoshan Xie, Guanghui Liu, Yongming Wu*, Shengnan Wang* Department of Neurology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Status epilepticus Immunostaining Immunotherapy Rat hippocampus section
Purpose: To evaluate the meaning of immunostaining of rat hippocampus with respect to clinical manifestations, Status epilepticus(SE)severity, treatment, and prognosis of New-Onset Refractory Status Epilepticus(NORSE) patients. Methods: Consecutive patients with cryptogenic NORSE admitted to the neuro-intensive care unit (NICU) between January 2015 and October 2018 were screened, and those who had serum and cerebrospinal fluid (CSF) immunostaining were included. Subsequently, the patients were classified into positive and negative immunostain groups. Immunotherapy was initiated in patients who progressed to super-RSE (SRSE). The demographic, and clinical, and immunostaining data were collected. The prognosis was evaluated by modified Rankin scale (mRS) at discharge (short-term prognosis) and 6 months (long-term prognosis), with mRS score ≤2 defined as the favorable outcome. The clinical manifestations, treatment response, and prognosis were compared between the patients with positive and negative immunostains. Results: 4/18 patients had positive immunostaining on both CSF and serum, 8 (had positive immunostaining on serum only, while 6 had negative immunostaining on serum and CSF. Twelve (66.7 %) patients progressed to SRSE, with no difference between the positive and negative immunostaining groups (P = 1.000). 7/18 patients had a favorable outcome at discharge and 11/18 after 6 months. No significant difference on the prognosis was detected between patients with positive and negative serum/CSF immunostaining and between the patients with or without immunotherapy at discharge or 6-month follow-up (P = 0.657/P = 0.502, P = 0.445/P = 0.829, P = 0.622, P = 0.567, respectively). Conclusions: The immunostaining on serum and/or CSF from NORSE patients did not indicate the progression to SRSE and clinical outcomes.
1. Introduction Status epilepticus (SE) is a life-threating neurological emergency which might be refractory to the available treatments [1]. Refractory SE (RSE) is used to describe the persistence of SE despite administration of at least 2 appropriately selected and dosed parenteral medications, including benzodiazepine [2]. New-Onset Refractory Status Epilepticus (NORSE) is often used in clinical practice to describe an adult patient who presents the RSE that remains unexplained after initial evaluation [2]. Cryptogenic NORSE is a subgroup of NORSE without a clear cause after extensive clinical workups [3].
Nowadays, epilepsy with autoantibodies against surface neuronal proteins has been recognized [4]. With the discovery of the antibodies, about 37 % of the NORSE was considered as antibody-mediated, while half was considered as cryptogenic [5]. For all the NORSE patients, if available, antibody test was suggested to differentiate the antibodymediated from cryptogenic. Systemic literature research suggested intensive and early immunotherapy for a patient with autoantibody-associated encephalopathy [6]. Therefore, for definite antibody-mediated NORSE, immunotherapy was reported to be promising. However, for the “seronegative” NORSE patients without specific autoantibodies, the importance of immunotherapy is pending [5,7].
⁎ Corresponding authors at: Department of Neurology, Nanfang Hospital, Southern Medical University, 1838 Northern Guangzhou Avenue, Guangzhou 510515, Guangdong, China. E-mail addresses: [email protected] (D. Wang), [email protected] (Y. Pan), [email protected] (K. Huang), [email protected] (Z. Lin), [email protected] (Z. Xie), [email protected] (G. Liu), [email protected] (Y. Wu), [email protected] (S. Wang). 1 Contributed equally.
https://doi.org/10.1016/j.seizure.2020.01.022 Received 8 December 2019; Received in revised form 22 January 2020; Accepted 30 January 2020 1059-1311/ © 2020 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.
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2.2. Data collection and analysis
Iizuka et al. reported eleven cryptogenic NORSE patients and found that 2 of the 10 treated patients respond to the first-line immunotherapy and 4 of the 5 patients respond to the second-line immunotherapy [5]. Li et al. reported three NORSE patients without definite etiology and all of them responded dramatically to the use of plasma exchange therapy with cessation of status epilepticus [8]. Additional evidence from other case series demonstrated successful treatment of NORSE by immunotherapy, indicating the underlying immune mechanism and the potential benefit of early immunotherapy [7,9]. Rat hippocampus immunostaining was performed on frozen rat brain sections [10] to visualize the neuron surface antibodies [11]. It is commonly used to identify the staining patterns of neuronal surface and synaptic proteins. The tests revealed subsets of patients, previously considered “seronegative,” who carried antibodies against the neuropil of brain [10]. However, the results should be confirmed by specific techniques [12]. Whether the positive result of the rat hippocampus section could be used to select the NORSE patients involved in the immune mechanism and subsequently be an indicator for immunotherapy was yet to be elucidated. Herein, we conducted a retrospective study to explore the results in the positive and negative immunostaining of rat hippocampus with respect to clinical manifestations, SE severity, treatment, regimen, and prognosis of NORSE patients. Thus, the immunostaining of rat hippocampus sections could be used as an indicator for immunotherapy in NORSE patients.
Demographic information and clinical data including CSF test results, autoimmune antibodies, MRI findings, rat hippocampus sections immunostaining, length of ICU stay, duration of ventilation, antiepileptic drug (AED) regimen, and immunotherapy were collected from the records. The immunotherapy included steroids, plasma exchange (PE), intravenous immunoglobin (IVIG), and immunosuppressant. All the patients were followed up in our clinic, and the prognosis was evaluated by modified Rankin scale (mRS) at the time of discharge (short-term prognosis) and follow-up at 6 months (long-term prognosis), with mRS score ≤2 defined as a favorable outcome. The data were analyzed using the SPSS 20.0 software (SPSS, IL, USA). The normally distributed continuous variables were summarized by mean and standard deviation, the non-normally distributed variables were represented as median, and categorical variables were expressed as absolute frequencies and percentages. The Mann–Whitney test or Chi-square test was used to analyze the non-normally distributed variables. All statistical tests were two-sided, and P-values < 0.05 were considered to be statistically significant. 2.3. Protocol of rat hippocampus section preparation and staining Rat hippocampal sections were prepared from sprague-dawley rat of either sex (weight: 200−300 g), in accordance with Institutional Animal Care and Use Committee–approved protocols. Rats were transcardially perfused with ice-cold normal saline. After perfusion, rats were decapitated and brains were sliced. The brain tissue was fixed by isopentane-liquid nitrogen and cut by the thickness of 5 μm. The slices with hippocampal tissue confirmed by hematoxylin-eosin staining were selected. During the staining procedure, 50 μL of tested sample was added to the section (serum dilution ratio 1:10, cerebrospinal fluid is not diluted), incubated at 37℃ for 1.5 h. 50 μL diluted delight488 labeled anti human IgG fluorescent antibody (dilution ratio: 1:200) was added and incubate at 37℃ for 1.5 h. Finally, the immunohistochemistry staining was completed and the section was evaluated under fluorescence microscopy.
2. Materials and methods 2.1. Patient selection In the present study, 20 consecutive patients with the diagnosis of cryptogenic NORSE [13] admitted to our neuro-intensive care unit (NICU) of Nanfang hospital, Guangzhou, China between January 1, 2015 and October 1, 2018, were reviewed in a retrospective study. All the patients underwent continuous video electroencephalography (cEEG) monitoring after admission. Finally, cryptogenic NORSE patients with serum and CSF immunostaining during hospitalization were included. The diagnosis of cryptogenic NORSE was based on the following criteria: 1) no history of epilepsy or seizures; 2) RSE: SE persisting despite administration of at least 2 appropriate parenteral medications including benzodiazepine [2]; 3) etiology remaining unknown after extensive workup as follows: Complete blood cell count (CBC), serum chemistry, thyroid function (free Triiodothyronine (FT3) and free Thyroxine (FT4) with thyroidstimulating hormone (TSH)), cerebrospinal fluid (CSF) for cell count, electrolytes, glucose, protein, cultures (bacterial, fungal); CSF viral polymerase chain reaction (herpes simplex virus, epstein-barr virus, varicella zoster virus, cytomegalovirus, human immunodeficiency virus, JC virus); CSF antibodies to screen for paraneoplastic intracellular antigens (CV2/CRMP5, Ma2, Ri, Yo, Hu, GAD65, and amphiphysin); CSF and serum neuron surface antibodies (NSAbs) to screen N-methyl-D-aspartate receptor (NMDAR), voltage gated potassium channel (VGKC), voltage gated calcium channel (VGCC), a-amino-3hydroxy-5- methyl-4-isoxazolepropionic acid receptor (AMPAR), gamino- butyric acid B receptor (GABABR), g-aminobutyric acid A receptor (GABAAR), metabotropic glutamate receptor (mGluR) 1, contactin-associated protein-like 2 (CASPR2), dipeptidyl peptidase-like protein 6 (DPP6), and leucine-rich glioma-inactivated 1 (LGI1); other serum autoantibodies (antinuclear antibody, anti-thyroid peroxidase (TPO)and anti-thyroglobulin (TG)); venereal disease research laboratory test (VDRL); CSF cytology; 3-Tesla brain magnetic resonance image (MRI) scan. This study was approved by the Ethics Committee of Nanfang Hospital, Southern Medical University. Informed consent was waived by the review board because this study was observational and retrospective, and all data were fully de-identified.
3. Results 3.1. Clinical characteristics A total of 20 patients diagnosed with cryptogenic NORSE were screened, and 2 cases did not undergo serum or CSF immunostaining during hospitalization. Finally, 18 patients were included in the analysis. The clinical characteristics of the cohort were demonstrated in Table 1. The median age of these patients was 23.5 (17–76) years. 12/ 18 patients were females. All the patients presented generalized tonicclonic seizure (GTCs) and received more than three types of anti-epilepsy medications (the AEDs used by the patients are listed in Table 3). A total of 12 (66.7 %) patients required mechanical ventilation (MV). The median duration of MV was 7 (0–72) days, and the median length of ICU stay was 25.5 (range, 2–126) days. The CSF examinations were conducted in all the patients within 24 h post-admission. The results revealed the mean opening pressure of lumbar puncture as 174.28 ± 15.04 mmH2O, the concentration of white blood cells was 5.39 ± 2.19 (0–8) cells/μL, the level of glucose was 4.00 ± 0.19 (normal range, 2.5–4.0) mmol/L, and the protein concentration was 0.37 ± 0.05 (normal range, 0.15–0.45) g/L. Thyroid function evaluation showed that median concentration of TSH was 1.283 (normal range: 0.55–4.78)mIU/L, the level of FT3 was 2.14 (normal range:2.3–4.2) pg/ml, and the level of FT4 was1.06 (normal range: 0.89–1.76)ng/dl. The screening of autoimmune antibodies showed weak positive antinuclear antibody (ANA) and slightly increased titers of anti-TG and 132
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Table 1 The clinical characteristics of the patients. Patient
Age
Gender
ICU duration (days)
Mechanical Ventilation (days)
Opening pressure (mmH2O)
CSF WBC (cells/ uL)
CSF protein (g/L)
CSF glucose (mmol/L)
ANA
TSH (mIU/ L)
FT3 (pg/ ml)
FT4 (ng/ dl)
Anti-TG (NR:0-115 U/L)
Anti-TPO (NR:0-34 U/L)
P1 P2 P3 P4 P5 P6 P7 P8 P9
68 20 76 38 19 58 24 22 24
Male Female Male Male Female Male Female Female Female
21 6 19 7 2 7 4 70 17
21 0 2 0 0 0.125 0 7 7
160 150 160 95 180 135 150 270 90
4 0 2 0 0 1 8 1 8
0.74 0.27 0.41 0.29 0.26 0.22 0.82 0.15 0.51
2.56 3.32 4.93 3.51 5.07 3.65 3.62 4.47 3.96
3.82 3.35 2.10 NA 0.72 0.95 0.53 1.28 0.76
2.91 2.67 1.71 NA 2.21 2.58 2.24 3.38 2.05
1.20 1.07 1.00 NA 0.93 1.06 1.10 1.24 1.33
12.2 14.05 < 10 NA < 10 25.32 15.95 32.69 63.39
14.13 11.81 18.54 NA 6.08 25.31 63.02 25.98 27.12
P10 P11 P12 P13
21 17 22 27
Female Female Female Female
32 30 53 103
11 19 12 61
150 165 260 170
0 10 0 36
0.14 0.27 0.22 0.37
4.07 4.25 4.47 3.00
0.58 6.43 2.33 2.08
1.12 2.14 3.13 2.13
0.86 0.92 1.31 0.96
118.8 169.5 35.87 108.3
82.57 33.25 26.83 21.88
P14
17
Female
19
0
80
7
0.42
3.79
0.76
2.14
0.88
< 10
9.29
P15 P16 P17 P18
27 62 23 23
Male Male Female Female
80 50 61 126
72 8 56 0
252 130 280 260
20 0 0 0
0.34 0.78 0.32 0.20
5.74 3.09 4.09 4.38
± ± Neg Neg Neg Neg Neg Neg Weak positive Neg Neg Neg Weak positive Weak positive Neg Neg Neg Neg
0.32 2.91 0.19 2.07
1.12 1.50 1.22 5.13
0.90 0.76 1.06 1.91
NA < 10 < 10 NA
NA 11.8 16.99 NA
Abbreviations: ANA: anti-nuclear antibody; CSF: cerebrospinal fluid; TG: thyroglobulin; TPO: thyroid peroxidase; FT3: free Triiodothyronine; FT4: free Thyroxine; TSH: thyroid-stimulating hormone; NA: not available; Neg: negative; NR: normal range; WBC: white blood cell.
with serum ANA positivity was observed. All the five patients with suspicious or weak-positive ANA showed positive serum rat hippocampus section immunostaining and 2 (40 %) showed positive CSF immunostaining. The comparison between patients with serum-positive and negative-immunostaining did not reveal any significant difference in thyroid functions including TSH (P = 0.132), FT3 (P = 0.234) and FT4 (P = 0.481).
anti-TPO in some cases without satisfying the diagnostic criteria of any connective tissue diseases or Hashimoto’s encephalopathy [14]. All the patients underwent an MRI scan with contrast if not contraindicated to exclude any occupying or cerebrovascular diseases. Strikingly, 4 cases revealed complete normal MRI, and 9 cases had slight abnormal signals in the cortex, subcortex, and periventricular white matter. Additionally, the meningeal enhancements were indicated in four cases, while temporal lobes were involved in three cases. Immunostaining using rat hippocampus section was conducted in all the patients (Ances et al.) [10] in both serum and CSF samples (Table 2). Of these patients, 4 (22.2 %) showed positive reactions for both serum and CSF, 8 (44.4 %) were positive for serum and negative for CSF, and six (33.3 %) were negative for both serum and CSF (Fig. 1). Intriguingly, none of the patients presented positive CSF and negative serum immunostaining. An overlap of serum-positive immunostaining
3.2. Treatment and outcome 12/18 patients received phenobarbital: 1 case received propofol and 17 cases received midazolam. A total of 12 (66.7 %) patients required immunotherapy, of which, 1 (8.3 %) needed an additional ketogenic diet for SE control and the other 2 (16.7 %) required mild intravenous hypothermia with muscle relaxant (rocuronium) and the ketogenic diet
Table 2 The immunohistochemistry tests of rat hippocampus section and cranial MRI findings of the patients. Patient
rat hippocampus section Serum
CSF
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16
Positive Positive Negative Positive Negative Positive Negative Positive Positive Negative Positives Negative Positive Positive Negative Positive
Negative Positive Negative Positive Negative Negative Negative Negative Positive Negative Negative Negative Negative Negative Negative Positive
P17 P18
Positive Positive
Negative Negative
MRI findings
Periventricular FLAIR high signals Normal Brain atrophy Diffused meningeal enhancements Normal Normal Diffused meningeal enhancements Slightly hyperintense signals of bilateral temporal lobes in FLAIR Patchy abnormal signals in bilateral frontal lobe, right parietal lobe, temporal lobe and insular lobe Diffused meningeal enhancements Patchy abnormal signals on the left parietal cortex and subcortex Normal Abnormal signals of bilateral cingulate gyrus, frontal and parietal gyrus Abnormal signals of bilateral temporal lobe and outer capsule with meningeal enhancement Multiple signal abnormalities in bilateral paraventricular and semioval centers Signal abnormalities in bilateral insular lobe, cerebral cortex and subcortical area, bilateral thalamus, lower cerebellar hemisphere and tonsil Normal Abnormal signals in bilateral basal ganglia and thalamus
Abbreviations: CSF: cerebrospinal fluid; FLAIR: fluid attenuated inversion recovery. 133
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Table 3 The treatment and outcome of the patients. Patient
AEDs and other anti-epilepsy treatment
Immunotherapy
mRS score at DC
mRS score at the 6th month
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11
Diazepam, Midazolam, VPA, LEV, OXC Diazepam, Midazolam, Propofol, LEV, LMT, VPA, OXC Diazepam, Midazolam, LEV, VPA, OXC Diazepam, LEV, Topiramate, Midazolam Diazepam, Midazolam, OXC, VPA, LEV Diazepam, Midazolam, VPA, PHB Diazepam, LEV, VPA, Midazolam Diazepam, Midazolam, VPA, LEV, PHB Diazepam, Midazolam, PHB, LEV, VPA Diazepam, Midazolam, PHB, OXC, VPA Diazepam, Midazolam, VPA, PHB, LEV, intravenous mild hypothermia, ketogenic diet Diazepam, Midazolam, PHB, OXC, LEV, VPA, Clonazepam Diazepam, Midazolam, Clonazepam, LEV, PHB Diazepam, Midazolam, PHB, VPA, LEV, Topiramate Diazepam, clonazepam, PHB, PhT, LEV, intravenous mild hypothermia, ketogenic diet Diazepam, Clonazepam, Midazolam, VPA, LMT, PHB, Topiramate Diazepam, VPA, LEV, PHB, Clonazepam, Topiramate, Midazolam Diazepam, LEV, LMT, OXC, Clonazepam, PHB, Midazolam, ketogenic diet
None None None MP 80 mg and taper None None IVIG, DXM 10 mg, taper to MP 12mg IVIG, MP pulse, PE IVIG IVIG,DXM 10 mg, PE IVIG, PE
6 1 5 5 0 5 2 5 1 2 2
6 1 4 2 0 2 0 2 1 0 2
IVIG, MP pulse, PE IVIG, MP 20 mg, PE IVIG, MP 120 mg, PE IVIG
5 4. 2 5
4 4 2 6
None IVIG, MP pulse, PE MP 40 mg, PE
5 3 5
6 1 5
P12 P13 P14 P15 P16 P17 P18
Abbreviations: AED: antiepileptic drug; DC: discharge; DXM: dexamethasone; LEV: levetiracetam; LMT, lamotrigine; IVIG, intravenous immunoglobin; MP: methylprednisolone; mRS: modified Rankin Scale; NA: not available; OXC, oxcarbazepine; PE: plasma exchange; PHB, phenobarbital; PhT: phenytoin sodium; VPA, valproate sodium.
results of serum hippocampus section immunostaining, 8/12 (66.7 %) patients of the positive group and 4/6 (66.7 %) patients of the negative group progressed to SRSE and received immunotherapy. The parentage of SRSE between the positive and negative groups did not differ
to control the SE. As a result, the SE was controlled in all patients with different anti-epilepsy regimens. In our unit, immunotherapy was initiated in patients with super refractory SE (SRSE) [2] who failed to be controlled after 24-h standard anti-epilepsy treatment. According to the
Fig. 1. Results of rat hippocampus section staining with serum or CSF. 1a showed the positive staining of rat hippocampus section. Patients’ serum or CSF present different staining types with cytoplasm-type (Fig. 1b) or cellular membrane-type (Fig. 1c). Fig. 1d showed the negative staining of rat hippocampus section. CSF, cerebrospinal fluid. 134
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NORSE transferring to SRSE between the serum-positive (66.7 %) and negative (66.7 %) groups was not significantly different, suggesting that the severity of NORSE was not associated with the serum immunostaining results. One possible explanation was that the severity of NORSE was not related to the antibodies. The other reason could be the limitation of immunostaining of rat hippocampus section [11]. Currently, the antibody-mediated neurological syndrome is reported, and the antibodies can be generally divided into two groups: antibodies targeting intracellular antigens and neuron surface antibodies [11]. Indirect immunostaining on frozen rat brain tissue is a preliminary screen, and the sensitivity depends on laboratory expertise [12]. Commercial assays for immunoblotting with recombinant proteins for the most common/well characterized paraneoplastic intracellular antibodies (Hu, Ma2, CV2/CRMP5, Ri, amphiphysin) are widely available. Also, in the case of patients with highly suspected NSAbs, commercial assays consisting of mosaics of cells displaying different NSAbs (for NMDAR, VGKC, VGCC, LGI1, CASPR2, AMPAR, GABAAR, GABABR, and GlyR) are available. Since the hippocampus section detected antibodies on the neuron surface in addition to intracellular antigens, the negative group might exhibit unknown intracellular antibodies. The detection of definite antibodies such as anti-NMDA receptor encephalitis and LGI1 on the cell surface or synaptic receptors prompt the initiation of immunotherapy, which might not be essential [11,15,16]. Moreover, for the cryptogenic NORSE patients without any specific autoantibodies, the importance of immunotherapy remains pending. Khawaja et al. reported 11 NORSE patients, of which 8 received any immunotherapy. 6/8 patients who received immunotherapy had favorable outcomes compared to none of the three patients who did not received any immunotherapy [7]. Other reported cases also showed that immunotherapy was beneficial in NORSE patients [7,9]. Patients with early immunotherapy showed favorable outcomes in 42 % as compared to the 20 % without the therapy [3,7,8,17]. In our center, when RSE evolved to be SRSE, immunotherapy was promptly initiated (12/18, 66.7 %). 7/18 (38.9 %) patients had a favorable outcome at discharge, and 61.1 % (11/18) showed the same at 6 months similar to the previous studies [7]. Furthermore, based on the current protocol, the patients with immunotherapy were more refractory than those without the therapy, which was attributed to the prolonged ICU stay and MV. Nevertheless, no significant difference was observed between the short-term and long-term prognosis of the patients with and without immunotherapy. Overall, there was no difference in prognosis between SRSE and RSE patients. The meaning of the result should be interpreted carefully. These differences might be offset by immunotherapy as SRSE patients received immunotherapy and RSE patients did not. Some cases showed that the prognosis of SRSE depends largely on the underlying etiology. Thus, it is vital to directly treat the underlying condition although most efforts are made to control the seizures [18]. Based on the present study on cryptogenic cases, when immunotherapy is initiated in SRSE patients, the patients with SRSE achieved prognosis as well as RSE. Moreover, the prognosis between the serum-positive and negative groups did not differ significantly. Also, SRSE patients with negative immunostaining might benefit from immunotherapy. The current results suggested that the initiation of immunotherapy in NORSE patients is based on the clinical manifestations in addition to the immunostaining results. In cryptogenic NORSE, brain MRIs showed symmetric T2/fluid-attenuated inversion recovery hyperintensities [19] and brain atrophy [5]. In this study, the MRI changes in the cryptogenic NROSE were not specific with scattered abnormal signals in the cortex or subcortex. Also, some patients presented complete normal MRI, while temporal lobe was involved in 3 cases. Moreover, enhanced meningeal changes were observed. Interestingly, whether these changes are purely due to the recurrent seizures or a part of underlying inflammatory disease is yet to be studied [4]. A majority of the MRI scans were performed when the seizures were successfully controlled. Since no definite cause was found, these changes were attributed to the seizures.
significantly (P = 1.000). The immunotherapy protocol varied according to the decisions of the neurologist, including methylprednisolone (MP) pulse therapy, a large dose of MP, IVIG), and PE (Table 3). The outcome of the patients was evaluated by mRS score at discharge and follow-up at 6 months. One patient expired because of cardiac arrest during hospitalization, 1 patient died of cardiac arrhythmia at 1-month post-discharge, and 1 died of severe infections at 1-week post-discharge. 7/18 (38.9 %) patients achieved favorable prognosis at discharge, and up to 61.1 % (11/18) of the patients achieved favorable prognosis at 6 months (Table 3). The comparison between patients with serum-positive and negativeimmunostaining did not reveal any significant difference in the shortterm and long-term prognosis (P = 0.657 and P = 0.445, respectively); also, no significant differences were detected with respect to the ICU stay and ventilation time (P = 0.542 and P = 0.924, respectively). Furthermore, the comparison between patients with positive and negative CSF immunostaining did not yield any significant difference in the short-term prognosis (P = 0.502), long-term prognosis (P = 0.829), length of ICU stay (P = 0.184), or duration of ventilation (P = 0.256). Additionally, the comparison between patients with immunotherapy (SRSE) or non-immunotherapy (non-SRSE) did not show any significant differences in the prognosis either at discharge or during the follow-up (P = 0.622 and P = 0.567, respectively). Thus, patients who received immunotherapy had a prolonged ICU stay (P = 0.061) and ventilation time (P = 0.390). In the sub-group of serum-positive patients, comparison between patients with SRSE and non-SRSE, no significant difference was observed in the prognosis either at discharge or during the follow-up (P = 0.447 and P = 0.374, respectively). 4. Discussion In this retrospective study, we reviewed 18 cryptogenic NORSE patients in our center. 12/18 (66.7 %) NORSE patients had positive serum immunostaining with or without positive CSF immunostaining. These patients progressed to SRSE without any difference between the positive and negative serum groups. 7/18 (38.9 %) patients exhibited favorable outcome at discharge, and 11/18 (61.1 %) showed the same at 6 months. No remarkable difference of prognosis was observed between the patients with positive and negative serum/CSF immunostaining at discharge or 6-month follow- up. No significant difference was detected in the prognosis between patients with or without immunotherapy. The current study differed from the previously reported cases such that the patients underwent examinations of rat hippocampus section immunostaining to visualize the neuron surface antibodies [11]. The test revealed subsets of patients, previously considered “seronegative,” who had antibodies against the neuropil of brain [10]. The patients who presented positive serum or CSF immunostaining were considered to have antibodies against hippocampus neuropils. Iizuka et al. reported 11 cryptogenic NORSE patients, and all were found to be negative for immunohistochemistry in rat brain or live hippocampus cultures. The study suggested that antibody-mediated mechanisms explained cryptogenic NORSE [5]. However, our result differed from that of the published cases with 66.7 % (12/18) serum-positive and 22.2 % (4/18) CSF-positive results, which supported the underlying immune mechanisms in NORSE. Especially, the overlap of serum ANA with positive serum hippocampus immunostaining was found in the study. The meaning of the concomitant antibodies was not clear and more research was required. Another interesting phenomenon was that all the CSFpositive cases had positive serum results and no serum-negative cases with positive CSF results, indicating that the antibody was probably transferred from the peripheral blood to the CSF other than intrathecal synthesis. 12/18 (66.7 %) cryptogenic NORSE patients progressed to be SRSE, consistent with the former studies which stated that NORSE is typically presented as super-refractory [2]. In this study, the percentage of 135
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laboratory which conducted the immunostaining of this study.
Nevertheless, the present study, consisting of the largest reported cryptogenic NORSE group, had some advantages. Also, the immune mechanisms underlying the cryptogenic NORSE were under intensive focus, and immunotherapy was recommended for the super refractory cases. The limitations of the study were as follows. Firstly, this was a retrospective single-center study. Secondly, paired case-control to evaluate the efficiency of immunotherapy and the rat hippocampus section immunostaining was lacking. Thirdly, as a cross-species reactivity test, the meaning of rat hippocampus section should be interpreted with caution. Finally, the sample size was small, and cryptogenic NORSE was an uncommon disease that accounted for about 10 % of SE [3].
References [1] Vezzani A, Dingledine R, Rossetti AO. Immunity and inflammation in status epilepticus and its sequelae: possibilities for therapeutic application. Expert Rev Neurother 2015;15:1081–92. [2] Hirsch LJ, Gaspard N, van Baalen A, Nabbout R, Demeret S, Loddenkemper T, Navarro V, Specchio N, Lagae L, Rossetti AO, Hocker S, Gofton TE, Abend NS, Gilmore EJ, Hahn C, Khosravani H, Rosenow F, Trinka E. Proposed consensus definitions for new-onset refractory status epilepticus (NORSE), febrile infection-related epilepsy syndrome (FIRES), and related conditions. Epilepsia 2018;59:739–44. [3] Costello DJ, Kilbride RD, Cole AJ. Cryptogenic New Onset Refractory Status Epilepticus (NORSE) in adults—infectious or not? J Neurol Sci 2009;277:26–31. [4] Irani SR, Bien CG, Lang B. Autoimmune epilepsies. Curr Opin Neurol 2011;24:146–53. [5] Iizuka T, Kanazawa N, Kaneko J, Tominaga N, Nonoda Y, Hara A, et al. Cryptogenic NORSE: its distinctive clinical features and response to immunotherapy. Neurol Neuroimmunol Neuroinflamm 2017;4:e396. [6] Bakpa OD, Reuber M, Irani SR. Antibody-associated epilepsies: clinical features, evidence for immunotherapies and future research questions. Seizure 2016;41:26–41. [7] Khawaja AM, DeWolfe JL, Miller DW, Szaflarski JP. New-onset refractory status epilepticus (NORSE) – the potential role for immunotherapy. Epilepsy Behav 2015;47:17–23. [8] Li J, Saldivar C, Maganti RK. Plasma exchange in cryptogenic new onset refractory status epilepticus. Seizure 2013;22:70–3. [9] Gall CRE, Jumma O, Mohanraj R. Five cases of new onset refractory status epilepticus (NORSE) syndrome: outcomes with early immunotherapy. Seizure 2013;22:217–20. [10] Ances BM, Vitaliani R, Taylor RA, Liebeskind DS, Voloschin A, Houghton DJ, Galetta SL, Dichter M, Alavi A, Rosenfeld MR, Dalmau J. Treatment-responsive limbic encephalitis identified by neuropil antibodies: MRI and PET correlates. Brain 2005;128:1764–77. [11] Lancaster E, Martinez-Hernandez E, Dalmau J. Encephalitis and antibodies to synaptic and neuronal cell surface proteins. Neurology 2011;77:179–89. [12] Zuliani L, Graus F, Giometto B, Bien C, Vincent A. Central nervous system neuronal surface antibody associated syndromes: review and guidelines for recognition. J Neurol Neurosurg Psychiatry 2012;83:638–45. [13] Brophy GM, Bell R, Claassen J, Alldredge B, Bleck TP, Glauser T, Laroche SM, Riviello Jr JJ, Shutter L, Sperling MR, Treiman DM, Vespa PM, Neurocritical Care Society Status Epilepticus Guideline Writing, C. Guidelines for the evaluation and management of status epilepticus. Neurocrit Care 2012;17:3–23. [14] Olmez I, Moses H, Sriram S, Kirshner H, Lagrange AH, Pawate S. Diagnostic and therapeutic aspects of Hashimoto’s encephalopathy. J Neurol Sci 2013;331:67–71. [15] Linnoila JJ, Rosenfeld MR, Dalmau J. Neuronal surface antibody-mediated autoimmune encephalitis. Semin Neurol 2014;34:458–66. [16] Varley J, Taylor J, Irani SR. Autoantibody-mediated diseases of the CNS: Structure, dysfunction and therapy. Neuropharmacology 2018;132:71–82. [17] Baxter P, Clarke A, Cross H, Harding B, Hicks E, Livingston J, et al. Idiopathic catastrophic epileptic encephalopathy presenting with acute onset intractable status. Seizure 2003;12:379–87. [18] Shorvon S. Super-refractory status epilepticus: an approach to therapy in this difficult clinical situation. Epilepsia 2011;52:53–6. [19] Cartagena AM, Young GB, Lee DH, Mirsattari SM. Reversible and irreversible cranial MRI findings associated with status epilepticus. Epilepsy Behav 2014;33:24–30.
5. Conclusions In this study, the immunostaining on serum and/or CSF from NORSE patients did not indicate any progression to SRSE and clinical outcome. Although immunotherapy seemed to benefit the NORSE patients progressed to SRSE, the therapeutic effect had no significant correlation with immunostaining results. Thus, large randomized clinical trials are essential to confirm these results. Authorship contributions DW, YP, KH, ZL, YW, SW are responsible for concepts and design. YP, KH are responsible for data collecting and statistical analysis. ZX and GL are responsible for immunostaining tests of all the patients. All authors contributed intellectually. All authors acquired, analyzed, and interpreted the data. The manuscript was prepared by DW, YW and SW. All authors reviewed and made critical revisions to the manuscript. Funding This study was supported by President Fund of Nanfang Hospital, Southern Medical University (No.2019B007). Declaration of Competing Interest The authors declared no conflicts of interest with respect to the research, authorship, funding, and/or publication of this article. Acknowledgements The authors thank Professor Yafang Hu in Neurology Department of Nanfang Hospital, Southern Medical University for managing the
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Is rat hippocampus section immunostaining an indicator for immunotherapy in cryptogenic adult new-onset refractory status epilepticus (NORSE)?
T
Dongmei Wang1, Yue Pan1, Kaibin Huang, Zhenzhou Lin, Zuoshan Xie, Guanghui Liu, Yongming Wu*, Shengnan Wang* Department of Neurology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Status epilepticus Immunostaining Immunotherapy Rat hippocampus section
Purpose: To evaluate the meaning of immunostaining of rat hippocampus with respect to clinical manifestations, Status epilepticus(SE)severity, treatment, and prognosis of New-Onset Refractory Status Epilepticus(NORSE) patients. Methods: Consecutive patients with cryptogenic NORSE admitted to the neuro-intensive care unit (NICU) between January 2015 and October 2018 were screened, and those who had serum and cerebrospinal fluid (CSF) immunostaining were included. Subsequently, the patients were classified into positive and negative immunostain groups. Immunotherapy was initiated in patients who progressed to super-RSE (SRSE). The demographic, and clinical, and immunostaining data were collected. The prognosis was evaluated by modified Rankin scale (mRS) at discharge (short-term prognosis) and 6 months (long-term prognosis), with mRS score ≤2 defined as the favorable outcome. The clinical manifestations, treatment response, and prognosis were compared between the patients with positive and negative immunostains. Results: 4/18 patients had positive immunostaining on both CSF and serum, 8 (had positive immunostaining on serum only, while 6 had negative immunostaining on serum and CSF. Twelve (66.7 %) patients progressed to SRSE, with no difference between the positive and negative immunostaining groups (P = 1.000). 7/18 patients had a favorable outcome at discharge and 11/18 after 6 months. No significant difference on the prognosis was detected between patients with positive and negative serum/CSF immunostaining and between the patients with or without immunotherapy at discharge or 6-month follow-up (P = 0.657/P = 0.502, P = 0.445/P = 0.829, P = 0.622, P = 0.567, respectively). Conclusions: The immunostaining on serum and/or CSF from NORSE patients did not indicate the progression to SRSE and clinical outcomes.
1. Introduction Status epilepticus (SE) is a life-threating neurological emergency which might be refractory to the available treatments [1]. Refractory SE (RSE) is used to describe the persistence of SE despite administration of at least 2 appropriately selected and dosed parenteral medications, including benzodiazepine [2]. New-Onset Refractory Status Epilepticus (NORSE) is often used in clinical practice to describe an adult patient who presents the RSE that remains unexplained after initial evaluation [2]. Cryptogenic NORSE is a subgroup of NORSE without a clear cause after extensive clinical workups [3].
Nowadays, epilepsy with autoantibodies against surface neuronal proteins has been recognized [4]. With the discovery of the antibodies, about 37 % of the NORSE was considered as antibody-mediated, while half was considered as cryptogenic [5]. For all the NORSE patients, if available, antibody test was suggested to differentiate the antibodymediated from cryptogenic. Systemic literature research suggested intensive and early immunotherapy for a patient with autoantibody-associated encephalopathy [6]. Therefore, for definite antibody-mediated NORSE, immunotherapy was reported to be promising. However, for the “seronegative” NORSE patients without specific autoantibodies, the importance of immunotherapy is pending [5,7].
⁎ Corresponding authors at: Department of Neurology, Nanfang Hospital, Southern Medical University, 1838 Northern Guangzhou Avenue, Guangzhou 510515, Guangdong, China. E-mail addresses: [email protected] (D. Wang), [email protected] (Y. Pan), [email protected] (K. Huang), [email protected] (Z. Lin), [email protected] (Z. Xie), [email protected] (G. Liu), [email protected] (Y. Wu), [email protected] (S. Wang). 1 Contributed equally.
https://doi.org/10.1016/j.seizure.2020.01.022 Received 8 December 2019; Received in revised form 22 January 2020; Accepted 30 January 2020 1059-1311/ © 2020 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.
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2.2. Data collection and analysis
Iizuka et al. reported eleven cryptogenic NORSE patients and found that 2 of the 10 treated patients respond to the first-line immunotherapy and 4 of the 5 patients respond to the second-line immunotherapy [5]. Li et al. reported three NORSE patients without definite etiology and all of them responded dramatically to the use of plasma exchange therapy with cessation of status epilepticus [8]. Additional evidence from other case series demonstrated successful treatment of NORSE by immunotherapy, indicating the underlying immune mechanism and the potential benefit of early immunotherapy [7,9]. Rat hippocampus immunostaining was performed on frozen rat brain sections [10] to visualize the neuron surface antibodies [11]. It is commonly used to identify the staining patterns of neuronal surface and synaptic proteins. The tests revealed subsets of patients, previously considered “seronegative,” who carried antibodies against the neuropil of brain [10]. However, the results should be confirmed by specific techniques [12]. Whether the positive result of the rat hippocampus section could be used to select the NORSE patients involved in the immune mechanism and subsequently be an indicator for immunotherapy was yet to be elucidated. Herein, we conducted a retrospective study to explore the results in the positive and negative immunostaining of rat hippocampus with respect to clinical manifestations, SE severity, treatment, regimen, and prognosis of NORSE patients. Thus, the immunostaining of rat hippocampus sections could be used as an indicator for immunotherapy in NORSE patients.
Demographic information and clinical data including CSF test results, autoimmune antibodies, MRI findings, rat hippocampus sections immunostaining, length of ICU stay, duration of ventilation, antiepileptic drug (AED) regimen, and immunotherapy were collected from the records. The immunotherapy included steroids, plasma exchange (PE), intravenous immunoglobin (IVIG), and immunosuppressant. All the patients were followed up in our clinic, and the prognosis was evaluated by modified Rankin scale (mRS) at the time of discharge (short-term prognosis) and follow-up at 6 months (long-term prognosis), with mRS score ≤2 defined as a favorable outcome. The data were analyzed using the SPSS 20.0 software (SPSS, IL, USA). The normally distributed continuous variables were summarized by mean and standard deviation, the non-normally distributed variables were represented as median, and categorical variables were expressed as absolute frequencies and percentages. The Mann–Whitney test or Chi-square test was used to analyze the non-normally distributed variables. All statistical tests were two-sided, and P-values < 0.05 were considered to be statistically significant. 2.3. Protocol of rat hippocampus section preparation and staining Rat hippocampal sections were prepared from sprague-dawley rat of either sex (weight: 200−300 g), in accordance with Institutional Animal Care and Use Committee–approved protocols. Rats were transcardially perfused with ice-cold normal saline. After perfusion, rats were decapitated and brains were sliced. The brain tissue was fixed by isopentane-liquid nitrogen and cut by the thickness of 5 μm. The slices with hippocampal tissue confirmed by hematoxylin-eosin staining were selected. During the staining procedure, 50 μL of tested sample was added to the section (serum dilution ratio 1:10, cerebrospinal fluid is not diluted), incubated at 37℃ for 1.5 h. 50 μL diluted delight488 labeled anti human IgG fluorescent antibody (dilution ratio: 1:200) was added and incubate at 37℃ for 1.5 h. Finally, the immunohistochemistry staining was completed and the section was evaluated under fluorescence microscopy.
2. Materials and methods 2.1. Patient selection In the present study, 20 consecutive patients with the diagnosis of cryptogenic NORSE [13] admitted to our neuro-intensive care unit (NICU) of Nanfang hospital, Guangzhou, China between January 1, 2015 and October 1, 2018, were reviewed in a retrospective study. All the patients underwent continuous video electroencephalography (cEEG) monitoring after admission. Finally, cryptogenic NORSE patients with serum and CSF immunostaining during hospitalization were included. The diagnosis of cryptogenic NORSE was based on the following criteria: 1) no history of epilepsy or seizures; 2) RSE: SE persisting despite administration of at least 2 appropriate parenteral medications including benzodiazepine [2]; 3) etiology remaining unknown after extensive workup as follows: Complete blood cell count (CBC), serum chemistry, thyroid function (free Triiodothyronine (FT3) and free Thyroxine (FT4) with thyroidstimulating hormone (TSH)), cerebrospinal fluid (CSF) for cell count, electrolytes, glucose, protein, cultures (bacterial, fungal); CSF viral polymerase chain reaction (herpes simplex virus, epstein-barr virus, varicella zoster virus, cytomegalovirus, human immunodeficiency virus, JC virus); CSF antibodies to screen for paraneoplastic intracellular antigens (CV2/CRMP5, Ma2, Ri, Yo, Hu, GAD65, and amphiphysin); CSF and serum neuron surface antibodies (NSAbs) to screen N-methyl-D-aspartate receptor (NMDAR), voltage gated potassium channel (VGKC), voltage gated calcium channel (VGCC), a-amino-3hydroxy-5- methyl-4-isoxazolepropionic acid receptor (AMPAR), gamino- butyric acid B receptor (GABABR), g-aminobutyric acid A receptor (GABAAR), metabotropic glutamate receptor (mGluR) 1, contactin-associated protein-like 2 (CASPR2), dipeptidyl peptidase-like protein 6 (DPP6), and leucine-rich glioma-inactivated 1 (LGI1); other serum autoantibodies (antinuclear antibody, anti-thyroid peroxidase (TPO)and anti-thyroglobulin (TG)); venereal disease research laboratory test (VDRL); CSF cytology; 3-Tesla brain magnetic resonance image (MRI) scan. This study was approved by the Ethics Committee of Nanfang Hospital, Southern Medical University. Informed consent was waived by the review board because this study was observational and retrospective, and all data were fully de-identified.
3. Results 3.1. Clinical characteristics A total of 20 patients diagnosed with cryptogenic NORSE were screened, and 2 cases did not undergo serum or CSF immunostaining during hospitalization. Finally, 18 patients were included in the analysis. The clinical characteristics of the cohort were demonstrated in Table 1. The median age of these patients was 23.5 (17–76) years. 12/ 18 patients were females. All the patients presented generalized tonicclonic seizure (GTCs) and received more than three types of anti-epilepsy medications (the AEDs used by the patients are listed in Table 3). A total of 12 (66.7 %) patients required mechanical ventilation (MV). The median duration of MV was 7 (0–72) days, and the median length of ICU stay was 25.5 (range, 2–126) days. The CSF examinations were conducted in all the patients within 24 h post-admission. The results revealed the mean opening pressure of lumbar puncture as 174.28 ± 15.04 mmH2O, the concentration of white blood cells was 5.39 ± 2.19 (0–8) cells/μL, the level of glucose was 4.00 ± 0.19 (normal range, 2.5–4.0) mmol/L, and the protein concentration was 0.37 ± 0.05 (normal range, 0.15–0.45) g/L. Thyroid function evaluation showed that median concentration of TSH was 1.283 (normal range: 0.55–4.78)mIU/L, the level of FT3 was 2.14 (normal range:2.3–4.2) pg/ml, and the level of FT4 was1.06 (normal range: 0.89–1.76)ng/dl. The screening of autoimmune antibodies showed weak positive antinuclear antibody (ANA) and slightly increased titers of anti-TG and 132
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Table 1 The clinical characteristics of the patients. Patient
Age
Gender
ICU duration (days)
Mechanical Ventilation (days)
Opening pressure (mmH2O)
CSF WBC (cells/ uL)
CSF protein (g/L)
CSF glucose (mmol/L)
ANA
TSH (mIU/ L)
FT3 (pg/ ml)
FT4 (ng/ dl)
Anti-TG (NR:0-115 U/L)
Anti-TPO (NR:0-34 U/L)
P1 P2 P3 P4 P5 P6 P7 P8 P9
68 20 76 38 19 58 24 22 24
Male Female Male Male Female Male Female Female Female
21 6 19 7 2 7 4 70 17
21 0 2 0 0 0.125 0 7 7
160 150 160 95 180 135 150 270 90
4 0 2 0 0 1 8 1 8
0.74 0.27 0.41 0.29 0.26 0.22 0.82 0.15 0.51
2.56 3.32 4.93 3.51 5.07 3.65 3.62 4.47 3.96
3.82 3.35 2.10 NA 0.72 0.95 0.53 1.28 0.76
2.91 2.67 1.71 NA 2.21 2.58 2.24 3.38 2.05
1.20 1.07 1.00 NA 0.93 1.06 1.10 1.24 1.33
12.2 14.05 < 10 NA < 10 25.32 15.95 32.69 63.39
14.13 11.81 18.54 NA 6.08 25.31 63.02 25.98 27.12
P10 P11 P12 P13
21 17 22 27
Female Female Female Female
32 30 53 103
11 19 12 61
150 165 260 170
0 10 0 36
0.14 0.27 0.22 0.37
4.07 4.25 4.47 3.00
0.58 6.43 2.33 2.08
1.12 2.14 3.13 2.13
0.86 0.92 1.31 0.96
118.8 169.5 35.87 108.3
82.57 33.25 26.83 21.88
P14
17
Female
19
0
80
7
0.42
3.79
0.76
2.14
0.88
< 10
9.29
P15 P16 P17 P18
27 62 23 23
Male Male Female Female
80 50 61 126
72 8 56 0
252 130 280 260
20 0 0 0
0.34 0.78 0.32 0.20
5.74 3.09 4.09 4.38
± ± Neg Neg Neg Neg Neg Neg Weak positive Neg Neg Neg Weak positive Weak positive Neg Neg Neg Neg
0.32 2.91 0.19 2.07
1.12 1.50 1.22 5.13
0.90 0.76 1.06 1.91
NA < 10 < 10 NA
NA 11.8 16.99 NA
Abbreviations: ANA: anti-nuclear antibody; CSF: cerebrospinal fluid; TG: thyroglobulin; TPO: thyroid peroxidase; FT3: free Triiodothyronine; FT4: free Thyroxine; TSH: thyroid-stimulating hormone; NA: not available; Neg: negative; NR: normal range; WBC: white blood cell.
with serum ANA positivity was observed. All the five patients with suspicious or weak-positive ANA showed positive serum rat hippocampus section immunostaining and 2 (40 %) showed positive CSF immunostaining. The comparison between patients with serum-positive and negative-immunostaining did not reveal any significant difference in thyroid functions including TSH (P = 0.132), FT3 (P = 0.234) and FT4 (P = 0.481).
anti-TPO in some cases without satisfying the diagnostic criteria of any connective tissue diseases or Hashimoto’s encephalopathy [14]. All the patients underwent an MRI scan with contrast if not contraindicated to exclude any occupying or cerebrovascular diseases. Strikingly, 4 cases revealed complete normal MRI, and 9 cases had slight abnormal signals in the cortex, subcortex, and periventricular white matter. Additionally, the meningeal enhancements were indicated in four cases, while temporal lobes were involved in three cases. Immunostaining using rat hippocampus section was conducted in all the patients (Ances et al.) [10] in both serum and CSF samples (Table 2). Of these patients, 4 (22.2 %) showed positive reactions for both serum and CSF, 8 (44.4 %) were positive for serum and negative for CSF, and six (33.3 %) were negative for both serum and CSF (Fig. 1). Intriguingly, none of the patients presented positive CSF and negative serum immunostaining. An overlap of serum-positive immunostaining
3.2. Treatment and outcome 12/18 patients received phenobarbital: 1 case received propofol and 17 cases received midazolam. A total of 12 (66.7 %) patients required immunotherapy, of which, 1 (8.3 %) needed an additional ketogenic diet for SE control and the other 2 (16.7 %) required mild intravenous hypothermia with muscle relaxant (rocuronium) and the ketogenic diet
Table 2 The immunohistochemistry tests of rat hippocampus section and cranial MRI findings of the patients. Patient
rat hippocampus section Serum
CSF
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16
Positive Positive Negative Positive Negative Positive Negative Positive Positive Negative Positives Negative Positive Positive Negative Positive
Negative Positive Negative Positive Negative Negative Negative Negative Positive Negative Negative Negative Negative Negative Negative Positive
P17 P18
Positive Positive
Negative Negative
MRI findings
Periventricular FLAIR high signals Normal Brain atrophy Diffused meningeal enhancements Normal Normal Diffused meningeal enhancements Slightly hyperintense signals of bilateral temporal lobes in FLAIR Patchy abnormal signals in bilateral frontal lobe, right parietal lobe, temporal lobe and insular lobe Diffused meningeal enhancements Patchy abnormal signals on the left parietal cortex and subcortex Normal Abnormal signals of bilateral cingulate gyrus, frontal and parietal gyrus Abnormal signals of bilateral temporal lobe and outer capsule with meningeal enhancement Multiple signal abnormalities in bilateral paraventricular and semioval centers Signal abnormalities in bilateral insular lobe, cerebral cortex and subcortical area, bilateral thalamus, lower cerebellar hemisphere and tonsil Normal Abnormal signals in bilateral basal ganglia and thalamus
Abbreviations: CSF: cerebrospinal fluid; FLAIR: fluid attenuated inversion recovery. 133
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Table 3 The treatment and outcome of the patients. Patient
AEDs and other anti-epilepsy treatment
Immunotherapy
mRS score at DC
mRS score at the 6th month
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11
Diazepam, Midazolam, VPA, LEV, OXC Diazepam, Midazolam, Propofol, LEV, LMT, VPA, OXC Diazepam, Midazolam, LEV, VPA, OXC Diazepam, LEV, Topiramate, Midazolam Diazepam, Midazolam, OXC, VPA, LEV Diazepam, Midazolam, VPA, PHB Diazepam, LEV, VPA, Midazolam Diazepam, Midazolam, VPA, LEV, PHB Diazepam, Midazolam, PHB, LEV, VPA Diazepam, Midazolam, PHB, OXC, VPA Diazepam, Midazolam, VPA, PHB, LEV, intravenous mild hypothermia, ketogenic diet Diazepam, Midazolam, PHB, OXC, LEV, VPA, Clonazepam Diazepam, Midazolam, Clonazepam, LEV, PHB Diazepam, Midazolam, PHB, VPA, LEV, Topiramate Diazepam, clonazepam, PHB, PhT, LEV, intravenous mild hypothermia, ketogenic diet Diazepam, Clonazepam, Midazolam, VPA, LMT, PHB, Topiramate Diazepam, VPA, LEV, PHB, Clonazepam, Topiramate, Midazolam Diazepam, LEV, LMT, OXC, Clonazepam, PHB, Midazolam, ketogenic diet
None None None MP 80 mg and taper None None IVIG, DXM 10 mg, taper to MP 12mg IVIG, MP pulse, PE IVIG IVIG,DXM 10 mg, PE IVIG, PE
6 1 5 5 0 5 2 5 1 2 2
6 1 4 2 0 2 0 2 1 0 2
IVIG, MP pulse, PE IVIG, MP 20 mg, PE IVIG, MP 120 mg, PE IVIG
5 4. 2 5
4 4 2 6
None IVIG, MP pulse, PE MP 40 mg, PE
5 3 5
6 1 5
P12 P13 P14 P15 P16 P17 P18
Abbreviations: AED: antiepileptic drug; DC: discharge; DXM: dexamethasone; LEV: levetiracetam; LMT, lamotrigine; IVIG, intravenous immunoglobin; MP: methylprednisolone; mRS: modified Rankin Scale; NA: not available; OXC, oxcarbazepine; PE: plasma exchange; PHB, phenobarbital; PhT: phenytoin sodium; VPA, valproate sodium.
results of serum hippocampus section immunostaining, 8/12 (66.7 %) patients of the positive group and 4/6 (66.7 %) patients of the negative group progressed to SRSE and received immunotherapy. The parentage of SRSE between the positive and negative groups did not differ
to control the SE. As a result, the SE was controlled in all patients with different anti-epilepsy regimens. In our unit, immunotherapy was initiated in patients with super refractory SE (SRSE) [2] who failed to be controlled after 24-h standard anti-epilepsy treatment. According to the
Fig. 1. Results of rat hippocampus section staining with serum or CSF. 1a showed the positive staining of rat hippocampus section. Patients’ serum or CSF present different staining types with cytoplasm-type (Fig. 1b) or cellular membrane-type (Fig. 1c). Fig. 1d showed the negative staining of rat hippocampus section. CSF, cerebrospinal fluid. 134
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NORSE transferring to SRSE between the serum-positive (66.7 %) and negative (66.7 %) groups was not significantly different, suggesting that the severity of NORSE was not associated with the serum immunostaining results. One possible explanation was that the severity of NORSE was not related to the antibodies. The other reason could be the limitation of immunostaining of rat hippocampus section [11]. Currently, the antibody-mediated neurological syndrome is reported, and the antibodies can be generally divided into two groups: antibodies targeting intracellular antigens and neuron surface antibodies [11]. Indirect immunostaining on frozen rat brain tissue is a preliminary screen, and the sensitivity depends on laboratory expertise [12]. Commercial assays for immunoblotting with recombinant proteins for the most common/well characterized paraneoplastic intracellular antibodies (Hu, Ma2, CV2/CRMP5, Ri, amphiphysin) are widely available. Also, in the case of patients with highly suspected NSAbs, commercial assays consisting of mosaics of cells displaying different NSAbs (for NMDAR, VGKC, VGCC, LGI1, CASPR2, AMPAR, GABAAR, GABABR, and GlyR) are available. Since the hippocampus section detected antibodies on the neuron surface in addition to intracellular antigens, the negative group might exhibit unknown intracellular antibodies. The detection of definite antibodies such as anti-NMDA receptor encephalitis and LGI1 on the cell surface or synaptic receptors prompt the initiation of immunotherapy, which might not be essential [11,15,16]. Moreover, for the cryptogenic NORSE patients without any specific autoantibodies, the importance of immunotherapy remains pending. Khawaja et al. reported 11 NORSE patients, of which 8 received any immunotherapy. 6/8 patients who received immunotherapy had favorable outcomes compared to none of the three patients who did not received any immunotherapy [7]. Other reported cases also showed that immunotherapy was beneficial in NORSE patients [7,9]. Patients with early immunotherapy showed favorable outcomes in 42 % as compared to the 20 % without the therapy [3,7,8,17]. In our center, when RSE evolved to be SRSE, immunotherapy was promptly initiated (12/18, 66.7 %). 7/18 (38.9 %) patients had a favorable outcome at discharge, and 61.1 % (11/18) showed the same at 6 months similar to the previous studies [7]. Furthermore, based on the current protocol, the patients with immunotherapy were more refractory than those without the therapy, which was attributed to the prolonged ICU stay and MV. Nevertheless, no significant difference was observed between the short-term and long-term prognosis of the patients with and without immunotherapy. Overall, there was no difference in prognosis between SRSE and RSE patients. The meaning of the result should be interpreted carefully. These differences might be offset by immunotherapy as SRSE patients received immunotherapy and RSE patients did not. Some cases showed that the prognosis of SRSE depends largely on the underlying etiology. Thus, it is vital to directly treat the underlying condition although most efforts are made to control the seizures [18]. Based on the present study on cryptogenic cases, when immunotherapy is initiated in SRSE patients, the patients with SRSE achieved prognosis as well as RSE. Moreover, the prognosis between the serum-positive and negative groups did not differ significantly. Also, SRSE patients with negative immunostaining might benefit from immunotherapy. The current results suggested that the initiation of immunotherapy in NORSE patients is based on the clinical manifestations in addition to the immunostaining results. In cryptogenic NORSE, brain MRIs showed symmetric T2/fluid-attenuated inversion recovery hyperintensities [19] and brain atrophy [5]. In this study, the MRI changes in the cryptogenic NROSE were not specific with scattered abnormal signals in the cortex or subcortex. Also, some patients presented complete normal MRI, while temporal lobe was involved in 3 cases. Moreover, enhanced meningeal changes were observed. Interestingly, whether these changes are purely due to the recurrent seizures or a part of underlying inflammatory disease is yet to be studied [4]. A majority of the MRI scans were performed when the seizures were successfully controlled. Since no definite cause was found, these changes were attributed to the seizures.
significantly (P = 1.000). The immunotherapy protocol varied according to the decisions of the neurologist, including methylprednisolone (MP) pulse therapy, a large dose of MP, IVIG), and PE (Table 3). The outcome of the patients was evaluated by mRS score at discharge and follow-up at 6 months. One patient expired because of cardiac arrest during hospitalization, 1 patient died of cardiac arrhythmia at 1-month post-discharge, and 1 died of severe infections at 1-week post-discharge. 7/18 (38.9 %) patients achieved favorable prognosis at discharge, and up to 61.1 % (11/18) of the patients achieved favorable prognosis at 6 months (Table 3). The comparison between patients with serum-positive and negativeimmunostaining did not reveal any significant difference in the shortterm and long-term prognosis (P = 0.657 and P = 0.445, respectively); also, no significant differences were detected with respect to the ICU stay and ventilation time (P = 0.542 and P = 0.924, respectively). Furthermore, the comparison between patients with positive and negative CSF immunostaining did not yield any significant difference in the short-term prognosis (P = 0.502), long-term prognosis (P = 0.829), length of ICU stay (P = 0.184), or duration of ventilation (P = 0.256). Additionally, the comparison between patients with immunotherapy (SRSE) or non-immunotherapy (non-SRSE) did not show any significant differences in the prognosis either at discharge or during the follow-up (P = 0.622 and P = 0.567, respectively). Thus, patients who received immunotherapy had a prolonged ICU stay (P = 0.061) and ventilation time (P = 0.390). In the sub-group of serum-positive patients, comparison between patients with SRSE and non-SRSE, no significant difference was observed in the prognosis either at discharge or during the follow-up (P = 0.447 and P = 0.374, respectively). 4. Discussion In this retrospective study, we reviewed 18 cryptogenic NORSE patients in our center. 12/18 (66.7 %) NORSE patients had positive serum immunostaining with or without positive CSF immunostaining. These patients progressed to SRSE without any difference between the positive and negative serum groups. 7/18 (38.9 %) patients exhibited favorable outcome at discharge, and 11/18 (61.1 %) showed the same at 6 months. No remarkable difference of prognosis was observed between the patients with positive and negative serum/CSF immunostaining at discharge or 6-month follow- up. No significant difference was detected in the prognosis between patients with or without immunotherapy. The current study differed from the previously reported cases such that the patients underwent examinations of rat hippocampus section immunostaining to visualize the neuron surface antibodies [11]. The test revealed subsets of patients, previously considered “seronegative,” who had antibodies against the neuropil of brain [10]. The patients who presented positive serum or CSF immunostaining were considered to have antibodies against hippocampus neuropils. Iizuka et al. reported 11 cryptogenic NORSE patients, and all were found to be negative for immunohistochemistry in rat brain or live hippocampus cultures. The study suggested that antibody-mediated mechanisms explained cryptogenic NORSE [5]. However, our result differed from that of the published cases with 66.7 % (12/18) serum-positive and 22.2 % (4/18) CSF-positive results, which supported the underlying immune mechanisms in NORSE. Especially, the overlap of serum ANA with positive serum hippocampus immunostaining was found in the study. The meaning of the concomitant antibodies was not clear and more research was required. Another interesting phenomenon was that all the CSFpositive cases had positive serum results and no serum-negative cases with positive CSF results, indicating that the antibody was probably transferred from the peripheral blood to the CSF other than intrathecal synthesis. 12/18 (66.7 %) cryptogenic NORSE patients progressed to be SRSE, consistent with the former studies which stated that NORSE is typically presented as super-refractory [2]. In this study, the percentage of 135
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laboratory which conducted the immunostaining of this study.
Nevertheless, the present study, consisting of the largest reported cryptogenic NORSE group, had some advantages. Also, the immune mechanisms underlying the cryptogenic NORSE were under intensive focus, and immunotherapy was recommended for the super refractory cases. The limitations of the study were as follows. Firstly, this was a retrospective single-center study. Secondly, paired case-control to evaluate the efficiency of immunotherapy and the rat hippocampus section immunostaining was lacking. Thirdly, as a cross-species reactivity test, the meaning of rat hippocampus section should be interpreted with caution. Finally, the sample size was small, and cryptogenic NORSE was an uncommon disease that accounted for about 10 % of SE [3].
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5. Conclusions In this study, the immunostaining on serum and/or CSF from NORSE patients did not indicate any progression to SRSE and clinical outcome. Although immunotherapy seemed to benefit the NORSE patients progressed to SRSE, the therapeutic effect had no significant correlation with immunostaining results. Thus, large randomized clinical trials are essential to confirm these results. Authorship contributions DW, YP, KH, ZL, YW, SW are responsible for concepts and design. YP, KH are responsible for data collecting and statistical analysis. ZX and GL are responsible for immunostaining tests of all the patients. All authors contributed intellectually. All authors acquired, analyzed, and interpreted the data. The manuscript was prepared by DW, YW and SW. All authors reviewed and made critical revisions to the manuscript. Funding This study was supported by President Fund of Nanfang Hospital, Southern Medical University (No.2019B007). Declaration of Competing Interest The authors declared no conflicts of interest with respect to the research, authorship, funding, and/or publication of this article. Acknowledgements The authors thank Professor Yafang Hu in Neurology Department of Nanfang Hospital, Southern Medical University for managing the
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