Expressions Of SMI-32 And CD-28K In Neocortex Of Cortical Dysplasia Model Rats

Objective: To investigate expressions of SMI-32 and CD-28K in neocortex of Cortical dysplasia(CD)model rats, so as to provide one new experimental method for pathogenesis study and diagnosis of CD.Methods: The twelve normal, healthy Sprague-Dawley(SD) rats (240-340g), eight- to twelve-week-old and on embryonic day 17(E17) were chosen at random and divided equally into two groups:①The first group(BCNU medicine test group), animals were given intraperitoneal injections with 15mg/kg of carmustine(1,3-bis-chloroethy 1-nitrosourea;BCNU) , BCNU was dissolved in sterile 5% dextrose in water at 4mg/l .②The control group, animals were injected with a corresponding volume of sterile 5% dextrose in water at the same day. All rats were fed normally in the same conditions and hoped for production. Pups were allowed to remain with their mother until P21 , at which time they were weaned.The results of ethology(including seizures) were observed and Morris-water-maze experiment was performed. The body weight and wet brain weight of pups were measured at P0,P28,andP56. (2) control and experimental rats (ages P28, P56) were anesthetized intraperitoneally. Sections were prepared from two contral group rats and two BCNU-exposed group rats at the age P28 and p56 .The rats were first transcardially perfused with 0.9% physiology saline and then with 4% paraformaldehyde in 0.01MPBS (PH=7.4). The brain was removed and post-fixed for 6-8 h in 4% paraformaldehyde in 0.01MPBS (PH=7.4), then was dehydrated in gradient at 4℃for 24 h (sink to the floor at best) with 20%,30% sucrose in 0.01MPBS(PH=7.4) in turn. Cryostat serial coronal sections including the primary somatosensory and motor cortices were obtained at 40-um thickness . Sections were chosen randomly for staining with Nissl staining (0.2%). Routine cortical histology was observed .Free floating sections staining was perfomed from sections chosen randomly by SP method so as to examine expression of SMI-32 and CD-28K in neocortex and the count ,morphology of SMI-32 and CD-28K positive cell .Results: (1)Litter size, ethology, and Moriss-water-maze experiment. No spontaneous seizure was observed in the fourty-five control and BCNUN-exposed litters.The pups in model group were poor than control group, followed by less activities, dullness and disturbance of intelligence in daily life.The escape latency of pups in model group was apparent longer than that of control group。BCNU-exposed pups had a significantly lower body and brain weight than those of controls (P0),the mean body weight was 7.5 vs 4.3 g and the mean wet brain weight was 372 vs 203 mg(P<0.05). By adult age (P56) Howere, control and BCNUN-exposed rats were found to have no significant difference in body weight. But the difference in mean brain wet weight was still significant.(1.81 vs. 1.23g, respectively ,p<0.05) . (2) Cortical histology. Examination of the brains of BCNU-exposed pups demonstrated obvious cortical maldevelopment. The entire quadrigeminal plate is visible on the dorsal surface of the brain in these animal. The brain weight of BCNU-exposed pups was reduced by ~40% .Nissl staining reveals significant differences in cortical architecture between BCNU-exposed and control cortex. Compared with the control cortex, the BCNU-exposed cortex shows a thin cortical plate and distinct clusters of neuronal elements that represent heterotopias. In the control adult rat cortex,the typical six-layered lamination pattern is apparent,whereas the BCNU-exposed cortex shows laminar disorgnization and thinning. Clusters of large neurons are in both superficial and deep layers in the BCNU-exposed cortex. (3) Immunohistochemistry①SMI-32 staining: demonstrated SMI-32 was expressed in layer II,III,V of the primary somatosensory cortex of control group rats. And expression was obvious,stable ,and stripped in layer V. In BCNU-exposed groups , the laminar architechture of cortex was disorganization , and the count of SMI-32 Positive cell was decreased ,and most of the SMI-32 Positive cell were shown in layer IV,VI ,but poorly in layerII,V. few were expressed in layer III. Pole orientation of cell was disorganized ,or lost. dendrite was aborized,cramped,and cell body was small with thin axual . sometimes dysmorphic,ball,or multipolar cell appeared. No matter control group or BCNU-exposed group rats, the size of the soma was larger but the CPC was less at P56 than that of at P28, and no change in distribution pattern of positive cells.②CD-28K staining: in the control groups rats , CD-28K labeled GABA inhibitoral interneurons in layer II/III,V/VI, but a lighter stained population was distributed throughout layers V/VI.The distribution of CD-28K positive cells were even and stripped..In the BCNU-treated rats,this distribution pattern was often disrupted and CD-28K-immunoreactive(ir) neurons were observed in differently shaped abnomal clusters,the count of CD-28K positive cell was decreased.Groups of CD-28k-ir neurons in the dorsal cortex were organized in radial columns or in nodules localized in the lowest part of the grey matter ,in the lateral cortex they were frequently localized in the superficial layers. P56,compared with P28,the size of soma was increased and no change on CPC and distribution pattern of positive cell in contol grouprats.P56,compared with P28,the size of soma was increased and the CPC was decreased and no change in distribution pattern of positive cell in contol group ratsConclusions: (1) Human CD can be simulated successfully by CD of BCNU-treated rats . (2) Routine histology staining (Nissl staining) can show cortical histology characteristics roughly but can't show origins of abnormal neurons and connections among neurons. SMI-32 and CD-28K staining may show abnormal cortical architecture and neurons for application to pathologic diagnosis of CD , and may also indicate etiopathogenesis of CD such as abnormalities of differentiation, migration, maturity and organization of neurons et al.