A Preliminary Study On The Morphology And Molecular Mechanism Of Cerebral Hernia Phenotype In Chickens

Cerebral hernia in crested chickens is characterized as the protuberance of cerebral hemispheres into the unsealed skull generally accompanied by cerebrospinal fluid.The abnormal development of cerebral hernia leads to abnormal behavior,low fertility and high mortality of animals,which will bring great economic losses to animal genetic breeding and reproduction.Although the appearance and behavior of chickens with cerebral hernia have been reported for hundreds of years since Charles Darwin,the mechanisms underlying the development and its effect on the brain remain largely unknown.This study provides anatomical and morphological descriptions that reveal the possible causes of cerebral hernia in crested chickens with or without cerebral hernia at 28 days after hatching.The telencephalon could be divided into different function regions by Nissl and hematoxylin and eosin stains to explore abnormal development of cerebral hernia effect on the structure of telencephalon functional regions and the cell state in chickens with or without cerebral hernia.Finally,we used to epigenetic approach to preliminarily explore the underlying mechanism of differential gene expression related to neurons and astrocytes.In conclusion,this study will preliminarily show the potential mechanism of cerebral hernia development in crested chickens,and provide theoretical bases and guidance to cerebral hernia development of crested chickens and other species.The main results are as follows:1.In chickens with cerebral hernia,the feathers on top of the head were elongated to form a high and upright crest compared with the short head feathers with no crest in chickens without cerebral hernia base on anatomical and morphological observation.Underneath the feather crest,the partial frontal skull was membranous lining that covered the telencephalon leading to the formation of an irregular skull hole.The telencephalon was not restricted by the continuous skull bone and protruded from the skull hole to form a protuberance accompanied by cerebrospinal fluid.Besides,the elongated telencephalon and other regions(the cerebellum and diencephalon)of the chicken brain with cerebral hernia were loosely united and separated from each other,rather than being closely packed together of the chicken brain without cerebral hernia.2.The location of the sphenoid bone that is positioned beneath and supports the telencephalon is approximately 45° from the horizontal line in chickens without cerebral hernia to a nearly vertical angle in chickens with cerebral hernia by the anatomy of the skull.The alteration of the sphenoid bone lifted the upper telencephalon.Hence,this abnormity of the sphenoid bone was possibly one of the causes of cerebral hernia.3.Nissl staining was used to detect the Nissl bodies inside the neurons of the telencephalon sagittal sections,and the telencephalon could be divided into seven regions(hyperpallium,hippocampus,mesopallium,nidopallium,Striatum,Pallidum,and Arcopallium)in chickens with or without cerebral hernia.Compared to chickens without cerebral hernia,Nissl staining and H&E staining revealed differential changes in shape and position with the misplaced pallidum,elongated and narrowed hippocampus,expanded mesopallium,nidopallium,and reduced hippocampus and hyperpallium.Nissl staining also revealed that the number of neurons was significantly decreased in H and Hp regions and increased in the M and N regions,respectively.Besides,the density of neurons was decreased in seven regions of the telencephalon in chickens with cerebral hernia,with the greatest reduction in the H,Hp,and P regions.4.We verified the mature neurons,neuron dendrites,and astrocytes by detecting the marker proteins Neu N,MAP2,and GFAP respectively,using immunohistochemistry to explore their differences.Neurons,neuron dendrites,and astrocytes were distributed evenly,clearly arranged and clear nucleus in the telencephalon tissue sections of chickens without cerebral hernia.We observed that neurons and astrocytes were disordered,intensely stained,among which number and density of neurons significantly reduced with shrinking of the neuron body,nucleus pyknosis and disorganization states neuron dendrites in chickens with cerebral hernia.5.The correlation between the expression level and methylation level of S100A6 and GFAP genes related to neurons and astrocytes were detected by bisulfite sequencing PCR(BSP).The down-regulation of DNA methylation levels in the promoter region of GFAP and S100A6 gene may lead to the up-regulation of GFAP and S100A6 gene expression in chickens with cerebral hernia.This suggests that epigenetic modification may be involved in the development of cerebral hernia,resulting in behavioral abnormalities of chickens with cerebral hernia.