A Study On Cognitive Deficits And Changes Of Cerebral Cholinergic Neurons Following Multiple Cerebral Concussion In Rats

Objective: To establish the model of multiple cerebral concussion (MCC) in rats, observe cognitive behavior, as well as the changes of cholinergic neurons in the basal forebrain, brainstem reticular formation, laterodorsal tegmental nucleus, dorsal nucleus of vagus and anterior horn cell of spinal cord post-MCC. Our aim is to study the relationship between cognitive deficits and the changes of cholinergic neurons after MCC in rats, thus to provide some preventive and treatment theories for sports-related cerebral concussion (SRCC) .Methods: Animal model of MCC in rats was developed using a metallic pendulum-striker concussive device. Forty-eight male Sprague-Dawley rats were randomly divided into six MCC groups (the group of 1d, 2d, 4d, 8d, 16d and 24d post-injury, n=8) . One control group was used(n=8) . After injury, vestibulomotor function and cognition were evaluated according to Beam Balance Test(BBT) and Beam Walking Test(BWT). Then, all animals were allowed to sacrifice and brains were removed. Frozen sections were stained with anti-ChAT(choline acetyltransferase) immunohistochemistry S-P method. The cholinergic neurons in the basal forebrain, brainstem reticular formation, laterodorsal tegmental nucleus, dorsal nucleus of vagus and anterior horn cell of spinal cord were observed and counted with optical microscope, and their expression was quantitated by image analysis system.Results: (1) Reduced latency on the BBT was observed in MCC rats on the first day post-injury (P<0．05 vs. control). Step through latency on the BWT prolonged conspicuously and persisted 10 days post-injury. With the exception of the ninth day, the statistical differences were significant compared with the control(P <0．05) . Score of BWT was significantly different up to 5 days post-injury (P <0．05 vs. control). (2) Compared with the control group, Loss of cholinergic neurons was observed in brain areas mentioned-above post-MCC. However, The statistical differences in different brain regions were diverse. That is, the significant loss of cholinergic neurons was examined from the first day to the 24^th day post-injury in the medial septal nucleus and dorsal nucleus of vertical limb diagonal band, the 4^th day post-injury in the left ventral nucleus of vertical limb diagonal band, the 4^th day and the 8^th day post-injury in the right ventral nucleus of vertical limb diagonal band, the 16^th day and 24^th day post-injury in the laterodorsal tegmental nucleus (P<0．05 vs. control). No loss of cholinergic neurons was detected in the nucleus of horizontal limb diagonal band, the basal nucleus of Meynert, brainstem reticular formation, dorsal nucleus of vagus and anterior horn cell of spinal cord.The positive expression of cholinergic neurons decreased significantly in the medial septal nucleus, ventral nucleus of vertical limb diagonal band and the level of pontine nucleus in brainstem reticular formation from the beginning of the 8^th day post-injury. Except for the left ventral nucleus of vertical limb diagonal band, others recovered to normal on the 24^th day post-injury. The expression of cholinergic neurons in the dorsal nucleus of vertical limb diagonal band attenuated markedly on 4^th day, then increased slightly, but dropped obviously again on 24^th day post-injury (P<0．05 vs. control). The expression of cholinergic neurons in the basal nucleus of Meynert, the level of trapezoid body and olivary nucleus in brainstem reticular formation was seriously lower than the control group on the 16^th day and 8^th day post-injury respectively. However, There were no significant difference in the nucleus of horizontal limb diagonal band, laterodorsal tegmental nucleus, dorsal nucleus of vagus and anterior horn cell of spinal cord compared with the control group. Conclusions: (1) The multiple cerebral concussion (MCC) model in rats was established successfully using a metallic pendulum-striker concussive device; ( 2 ) MCC created reversible and different types of vestibulomotor dysfunction and cognitive deficits in rats; ( 3 ) The decreasing tendency of the cholinergic neurons and their expression was observed in the basal forebrain, brainstem reticular formation, laterodorsal tegmental nucleus, dorsal nucleus of vagus and anterior horn cell of spinal cord following MCC in rats, especially serious on the 8^th day and 16^th day post-injury; (4) The loss of cholinergic neurons and their low expression in brain maybe one of the important factors of cognitive deficits post-MCC in rats.