There were 16 males and 10 females in the RE group, with a mean age of seizure onset of 5.8 years and a mean age at surgery of 7.5 years. Notably, about half of these had a definite preceding infection history. The 16 patients in the control group were generally matched with the RE patient group for age.
All clinical signs and treatments for patients with RE were described previously (Zhang et al., 2017). In our study, all RE patients were diagnosed according to clinical signs, histopathological changes in brain tissue, and magnetic resonance imaging (MRI) of the brain. All patients showed epilepsia partialis continua (EPC) that did not respond to antiepileptic drugs. According to HE staining, typical pathological characteristics were present in RE brain tissues, such as neuronal loss, neuronophagia (Figure 1A), microglial and lymphocytic nodules (Figure 1B), and perivascular cuffing (Figure 1C). According to MRI, unihemispheric focal cortical atrophy was observed to varying degrees, and most patients (18/26 cases) showed unilateral enlargement of the ventricular system and T2/FLAIR hyperintense signal in cortical or subcortical regions (Figure 1D–1F). These clinical signs, pathological changes, and MRI results support the diagnosis of RE.
Figure 1. MRI characteristics and pathological changes in the brains of RE patients. Typical pathological changes of RE were observed, including neuronophagia (A, scale bar = 50 μm), microglial and lymphocytic nodules (B, scale bar = 50 μm), and perivascular cuffing (C, scale bar = 100 μm). Atrophy of the right hemisphere cortex (arrows) and widening of the sulcus and caudate nucleus (arrowhead) were determined based on T1, T2, and FLAIR with hyperintense signal (D, E, and F). Brain atrophy confined to a single cerebral hemisphere is the most characteristic MRI feature of RE.
Expression of EBV in the brain tissues of RE patients or controls was analyzed by IHC using an antibody against the latent membrane protein 1 (LMP1) of EBV. The results were evaluated according to a scoring methodology described previously (Allred et al., 1998; Wang et al., 2003). In agreement with the previous report, expression of EBV was significantly higher in the RE group than in the control group, as shown in Table 1 and Figure 2B. Among the 26 RE cases, antigens of EBV were detected in 14 cases (53.8%). Two cases exhibited strong and moderate positive staining, while the other 12 cases showed weakly positive staining. In the control group, all 16 cases were negative for EBV staining. EBV antigen was located in the cytoplasms and nuclei of neuron-like cells in RE lesion areas (Figure 2A), indicating the involvement of EBV in RE.
No. cases (%) Total +++ ++ + RE (n = 26) 1 (3.85) 1 (3.85) 12 (46.15) 14 (53.85) Control (n = 16) 0 (0) 0 (0) 0 (0) 0 (0) Note: +++: Strongly positive; ++: Moderately positive; +: Weakly positive.
Table 1. Expression of EBV antigen in brain tissues of RE patients
Figure 2. EBV expression in brain tissues of RE patients and controls. (A) Representative images of strong, moderate, and weak positive staining and negative staining for EBV antigen under low (scale bar = 100 μm) and high (scale bar = 50 μm) magnification. Neuron-like cells were stained. (B) Percentages of RE patients and controls with strong, moderate, weak, or no EBV expression. (C) Analysis of correlation between EBV infection and brain atrophy. Atrophy grade (AG) was scored on a scale from 0 to 3 (Zhang et al., 2017), with higher scores indicating more severe atrophy. All EBV-positive cases showed brain atrophy to varying degrees. In contrast, there were eight cases with an AG score of 0 among 12 EBV-negative cases. EBV infection and brain atrophy were thus significantly associated (P = 0.001).
Next, the correlation between EBV infection and AG was analyzed. In this study, elevated expression of EBV antigen was observed in the RE group, and RE patients with positive EBV staining exhibited more severe brain atrophy (Table 2, Figure 2C) than EBV-negative cases (P < 0.05). This result suggests that EBV infection is associated with RE disease progression.
Brain atrophy Non-atrophy Total EBV-positive cases 14 0 14 EBV-negative cases 4 8 12 Total 18 8 26 Note: Fisher’s exact test: P = 0.001.
Table 2. Association between brain atrophy and EBV antigen expression in brain tissues of RE patients
According to IHC, positive staining for both TLR3 and TLR9 was observed in all RE cases (100%), while only six individuals (37.5%) with weakly positive staining were observed in the control group (Table 3). Interestingly, there were different staining intensities for TLR3 and TLR9 among RE brain specimens. For TLR3, strong staining was observed in 14 (23.8%) cases, and the other 12 cases (46.2%) showed weak staining. For TLR9, strong staining was observed in eight cases (30.8%), while the other 18 cases (69.2%) showed weak staining. Accordingly, highly expression of TRIF, a downstream molecule of TLR3, was also detected in the brain tissues of 14 RE patients (53.8%), 15.3% with strong expression and 38.5% with weak expression. In contrast, there were only six individuals (37.5%) weakly positive for TRIF staining in the control group. TLR3 and TLR9 were highly expressed in the cytoplasms of neurons and astrocytes, while TRIF was detected in both the cytoplasms and nuclei of neurons and astrocytes (Figure 3A). The above results indicate that TLR3, TLR9, and TRIF were clearly upregulated in the RE group (Figure 3B).
No. cases (%) Total +++/++ + TLR3 RE 14 (53.85) 12 (46.15) 26 (100) Control 1 (6.25) 5 (31.25) 6 (37.5) TLR9 RE 8 (30.77) 18 (69.23) 26 (100) Control 2 (12.5) 4 (25) 6 (37.5) TRIF RE 4 (15.38) 10 (38.46) 14 (53.85) Control 2 (12.5) 4 (25) 6 (37.5) Note: RE group: n = 26; Control group: n = 16; Strongly positive: +++/++; Weakly positive: +.
Table 3. Expression of TLRs in brain tissues of RE patients
Figure 3. TLR3, TLR9, and TRIF expression in brain tissues of RE patients and controls. (A) Representative images of TLR3, TLR9, and TRIF expression in neurons and astrocytes derived from brain tissues of RE patients under low (scale bar = 100 μm) and high (scale bar = 50 μm) magnification. (B) Percentages of RE patients and controls with strong, weak, or no expression of TLR3, TLR9, and TRIF. (C) Analysis of correlation between TLR3 expression and brain atrophy.
Moreover, we analyzed the correlation between TLR3 expression and the AG of the brain (Table 4, Figure 3C). Among the 26 RE patients, 14 RE cases with high expression of TLR3 exhibited clear brain atrophy, with AG scores of 2–3. In addition, among the remaining 12 RE individuals, six cases with weak staining of TLR3 showed no atrophy. The evidence therefore indicates that TLR3 expression is somewhat associated with RE progression.
Brain atrophy cases Non-atrophy cases Total +++/++ 12 2 14 + 6 6 12 Total 18 8 26 Note: Strongly positive: +++/++; Weakly positive: +; Fisher’s exact test: P = 0.090.
Table 4. Association between brain atrophy and TLR3 expression levels in brain tissues of RE patients
Elevated expression of EBV and TLRs in the brain is associated with Rasmussen’s encephalitis
- Received Date: 26 July 2017
- Accepted Date: 30 September 2017
- Published Date: 30 October 2017
Abstract: Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, the etiology of which remains unclear. It has been speculated that the immunopathogenesis of RE involves damage to neurons, which eventually leads to the occurrence of RE. Viral infection may be a critical factor in triggering RE immunopathogenesis. In this study, we analyzed the expression of Epstein-Barr virus (EBV) antigens as well as of Toll-like receptor 3 (TLR3), TLR9, and downstream adapter TIRdomain-containing adapter-inducing interferon-β (TRIF) in the brain tissues of 26 patients with RE and 16 control individuals using immunohistochemistry (IHC). In the RE group, EBV antigens were detected in 53% of individuals at various expression levels. In contrast, there was no detectable EBV antigen expression in control brain tissues. Moreover, we found marked increases in the expression of TLR3, TLR9, and TRIF in the brain tissues of RE patients compared with levels in the control group. Furthermore, among RE cases, EBV expression and high TLR3 expression were associated with more severe brain atrophy. Our results suggest that the elevated expression of EBV and TLRs may be involved in RE occurrence through the activation of downstream molecules.