Clemastine promotes recovery of neural function and suppresses neuronal apoptosis by restoring balance of pro-inflammatory mediators in an experimental model of intracerebral hemorrhage

Abstract

Intracerebral hemorrhage (ICH) represents a common acute cerebrovascular event that imparts high rates of disability. The microglia-mediated inflammatory response is a critical factor in determining cerebral damage post-ICH. Clemastine (CLM) is a histamine receptor H1 (HRH1) antagonist that has been shown to modulate the inflammatory response. However, the effects of CLM on ICH and the underlying mechanism remain to be determined. This investigation reveals that CLM resulted in reduction of cerebral hematoma volume, decreased cerebral edema and lower rates of neuronal apoptosis as well as improved behavioral scores in an acute ICH murine model. CLM treatment was noted to decrease pro-inflammatory effectors and increased anti-inflammatory effectors post-ICH. In addition, CLM reduced the deleterious effects of activated microglia on neurons in a transwell co-culture system. Our findings show that CLM likely mediates its therapeutic effect through inhibition of microglia-induced inflammatory response and apoptosis, thereby enhancing restoration of neuronal function.

Keywords: Clemastine; Intracerebral hemorrhage; histamine receptor H1.

Figure 1

Intraperitoneal injection of CLM restored or accelerated neurological function. (A) Garcia test scores of mice cohorts at 24, 48 and 72 hours post-ICH or sham-operation. (B) Coronal sections show lesion areas of mice brains belonging to three different groups (Left), and quantitative analyses of the lesion volumes 72h (n = 5, each group) post-ICH(Right). (C) Cerebral water content of the ipsilateral hemorrhagic hemispheres was assessed at 72h (n = 5, each group) post-ICH in each group. (D) Percentage of apoptotic cells after 72h (Bar = 50 µm). *p<0.05, **p<0.01, ***p<0.001, in contrast to the sham group; #p<0.05, #in contrast to the ICH + vehicle group.

Figure 2

The anti-inflammatory effect of CLM in peri-hemorrhagic areas. Inflammatory effectors iNOS, Agr-1, TNF-α and IL-1β were detected at gene and protein levels at 72h by RT-PCR (A) and western blotting (B). (C) Quantification of iNOS and Agr-1 protein expressions in the lesions after 72 h (scale bar = 50 µm). *p<0.05, in contrast to the sham group; #p<0.05, in contrast to the ICH + vehicle group.

Figure 3

CLM reduced RBC lysis-induced microglial activation in vitro. iNOS, Arg-1, TNF-α and IL-1β were detected at gene and protein levels at 72h by RT-PCR (A) and Western blotting. (B) Microglial cells co-cultured with primary neurons in the presence of lysed RBCs and treated with CLM. *p<0.05, in contrast to the sham group; #p<0.05, in contrast to the ICH + vehicle group.

Figure 4

CLM reduced neuronal damage induced by RBC lysis in vitro. (A) A schematic diagram of microglial cells co-cultured with primary neurons in the presence of lysed RBCs using transwell inserts. (B) Images of NeuN+ (green) primary neurons co-cultured and treated as described (scale bar = 50 µm) and their viability (C). *p<0.05, in contrast to the sham group; #p<0.05, in contrast to the ICH + vehicle group.