Upregulations of CRH and CRHR1 in the Epileptogenic Tissues of Patients with Intractable Infantile Spasms

Abstract

Aim: Infantile spasms (IS) are an age-specific epileptic syndrome with specific clinical symptom and electroencephalogram (EEG) features, lacking treatment options, and a poor prognosis. Excessive endogenous corticotropin-releasing hormone (CRH) in infant brain might result in IS. However, the data from human IS are limited. In our study, we investigated the expressions of CRH and its receptor type 1 (CRHR1) in surgical tissues from patients with IS and autopsy controls.

Methods: Specimens surgically removed from 17 patients with IS, and six autopsy controls were included in the study. Real-time PCR, Western blotting, and immunostaining were used to detect the expressions of mRNA, protein expression, and distribution. The correlation between variates was analyzed by Spearman rank correlation.

Results: The expressions of CRH and CRHR1 were significantly upregulated in the epileptogenic tissues of IS patients compared with the control group. CRH was distributed mainly in neurons, while CRHR1 was distributed in neurons, astrocytes, and microglia. The expression levels of CRH and CRHR1 were positively correlated with the frequency of epileptic spasms. Moreover, the expression of protein kinase C (PKC), which was an important downstream factor of CRHR1, was significantly upregulated in the epileptogenic tissues of patients with IS and was positively correlated with the CRHR1 expression levels and the frequency of epileptic spasms.

Conclusion: These results suggest that the CRH signal transduction pathway might participate in the epileptogenesis of IS, supporting the hypothesis that CRH is related to the pathogenesis of IS.

Keywords: Corticotrophin-releasing hormone receptor 1 (CRHR1); Corticotropin-releasing hormone (CRH); Infantile spasms (IS); Protein Kinase C (PKC).

Figure 1

Real‐time PCR of CRH and CRHR1mRNA expressions in control cortexes (n = 5) and infantile spasms (IS) specimens (n = 5). Messenger RNA level for CRH (A) and CRHR1 (B) was significantly upregulated in IS specimens compared to Controls. Respectively *P = 0.037, *P = 0.019, independent‐samples T test. (C–F) Alteration of corticotropin‐releasing hormone (CRH) and corticotropin‐releasing hormone receptor 1 (CRHR1) expression in the epileptogenic tissues of IS. (C–F) Representative immunoblot bands (C, E) and densitometric analysis (D, F) of total homogenates from IS specimens and Controls. CRH and CRHR1 are significantly increased in the epileptogenic tissues of IS compared to Controls. Respectively **P < 0.001, *P = 0.038, independent‐samples T test.

Figure 2

Representative hematoxylin and eosin staining photomicrographs of FCD Ia in infantile spasms (IS) samples. (A) FCD Ia samples showing cortical disorganization with the presence of microcolumns. (B–L) Immunoreactivity (IR) of CRH in epileptogenic tissues of IS and Controls. Weak CRH IR in neurons of normal cortex (arrows in B) and moderate to strong CRH IR in normal‐appearing neurons of epileptogenic tissues of IS (arrows in C). Co‐localization of CRH (green) with NF200 (red) in normal‐appearing neurons (arrows in DF). No CRH positive (green) astrocytes co‐localized with GFAP (red) (arrows in G–I), No CRH positive (green) microglia co‐localized with HLA (red) (arrows in J–L). Scale bars, 50 μm for A–C panels, 25 μm for D–L panels.

Figure 3

Representative hematoxylin and eosin staining photomicrographs of the normal‐appearing cortexes in infantile spasms (IS) samples (A). (B–L) Immunoreactivity (IR) of CRHR1 in epileptogenic tissues of IS and Controls. Weak to moderate CRHR1 IR in neurons (arrows in B) and glial cells (head arrows in B) in Control, Moderate to strong CRHR1 IR in normal‐appearing neurons (arrows in C) and glia cells (head arrows in C) in epileptogenic tissues of IS. Double‐labeled immunofluorescence shows co‐localization of CRHR1 (green) with NF200 (red) in normal‐appearing neurons (arrows in D–F). CRHR1 positive (green) astrocytes co‐localized with GFAP (red) (arrows in G–I), CRHR1 positive (green) microglia (arrows) co‐localized with HLA (red) (arrows in J–L). Scale bars, 50 μm for A–C panels, 25 μm for D–L panels, 10 μm for L–N panels.

Figure 4

Alteration of Protein Kinase C (PKC) in epileptogenic tissues of infantile spasms (IS). (A, B) Representative immunoblot bands (A) and densitometric analysis (B) of total homogenates from control cortexes and IS specimens. PKC are significantly increased in epileptogenic tissues of IS compared to Controls. **P < 0.001, independent‐samples T test. (C–N) Cell specific distribution of PKC in Controls and IS specimens. Representative hematoxylin and eosin staining photomicrographs of atypical gliosis in IS samples (C). Weak to moderate PKC Immunoreactivity (IR) in neurons (arrows in D) and glia cells (headarrows in D) of control cortex, moderate to strong PKC IR in normal‐appearing neurons (arrows in E) and glia cells (headarrows in E) of epileptogenic tissues of IS. Double‐labeled immunofluorescence shows co‐localization of PKC (green) with NF200 (red) in normal‐appearing neurons (arrows in F–H). PKC positive (green) astrocytes co‐localized with GFAP (red) (arrows in I–K), PKC positive (green) microglia (arrows) colocalized with HLA (red) (arrows in L–N), neuron do not co‐localized with HLA(red) (arrowhead in L, N). Scale bars, 50 μm for C–E panels, 25 μm for F–H panels, 10 μm for I–N panels.

Figure 5

(A–C) Correlation between the expression of CRH, CRHR1 and PKC and the frequency of epileptic spasm of IS. Scatter plot showing the significant positive correlation between the expression (relative optical density [OD]) of CRH, CRHR1 and PKC and seizure frequency (seizures per month) of IS. Spearman rank correlation coefficient: CRH: r = 0.671, P < 0.05; CRHR1, r = 0.689, P < 0.05; PKC, r = 0.72, P < 0.05. (D) Correlation between the expression of CRHR1 and PKC in IS. Scatter plot showing the significant positive correlation between the expression (relative optical density [OD]) of PKC and the expression of CRHR1. Spearman rank correlation coefficient r = 0.855, P < 0.01.