The Classification Standard Of Mild And Moderate Brain Injury In Rats, Cognitive Behavioral Disorder Pathology And Cholinergic Nerve Mechanism Study

Objective To set up a graded standard of mild and moderate traumatic brain injury (TBI) . To observe the changes of spatial cognition, sensorimotor faculties and retrograde amnesia, histopathology and brain ultrastructure, cholinergic neuron and its receptors in M₁, M₂ and N following brain concussion(BC) in rats. Methods The mild and moderate TBI rats model was developed by a metallic pendulum-striker device. Whole study included four parts. Firstly, after injury , five variables, that is, the time of apnea and the areflexia time of corneal reflex, external auditory canal stung reaction, body-righting reflex and needling reaction were actually direct measured and scored by a rat coma scoring standards, thus to set up a multivariate discriminant coefficient. Secondly, the spatial cognition was assessed by Morris Water Maze(MWM). The retrograde amnesia(RA) was assessed from 3 days of preinjury to 7 days of postconcussion by MWM Test. Beam Balance Test (BBT) latency was measured on days 1-5 after injury to assess vestibulomotor function. Step Through Latency and score on Beam Walking Test(BWT) were measured on days 1-5 after injury to evaluate coordinated walking ability, motion learning behavior. Thirdly, after sacrifice on time, paraffin sections were stained with HE, Nissl and silver, the brain ultrastructure of frontal contex, thalamas and brain stem were examined with a transmission electron microscope (TEM) . Forthly, the changes of cholinergic neuron and M₁, M₂ and N acetylcholine receptors were studied by ChAT antibody, MiR antibody, M₂R anbibody and NR antibody immunostain. Results (1) The probability of mild and moderate TBI distinguished according to actual direct measured value was 88.9%. The probability of mild and moderate TBI distinguished according to the rat coma scoring standards was 91.9%. (2) Cognitive behavior:(1)Reduced latency on MWM was observed in PCC rats(p<0. 05. vs control) up to first 6 days post injury and in CCC rats(p<0. 05. vs control)up to first 11 day post injury. (2) Compared with the control group, the retrograde amnesia was detected first 3 days in PCC groups, and first 5 days in CCC group after injury. At the same time, the both groups manifested space learn deficit. ?Reduced latency on the BBT was observed in CCC rats (P<0. 05 vs. control) up to first 5 days post-injury, however, PCC did not differ from Control. In the PCC group rats, motor and equilibrium function deficits in BWT were observed in the first 2 days (P<0. 05 vs. control) , Whatever, CCC groups persisted deficiency for first 5 days after injury. (3)Histopathological changes: The degeneration and necrosis of neuron and axonal injury were observed in cortex, hippocampus, hypothalamus, cerebrum bottom area, brain stem and cerebellum. Those pathological changes showed two injury peaks at 2 and 8-16 days after trauma.(4) Ultrastructure changes: The swelling and pyknosis in neurons and neuroglia cell, myelin sheath segregating and axons soluting were observed in front cortex, thalamus and brainstem. With the lapse of time, these changes aggravated gradually up to 8-16d post-injury,while capillaries were squeezed to occlude due to serious edema of astrocytes around it or platelet thrombus emerged. Then, the pathological changes lessened bit by bit. However, axonal injury, hyperplasia of glial cells and microcirculation disorder still existed to 64d after injury.(5) Cholinergic neurons changes:(1)the cholinergic cell numbers and the ChAT expression activity of cholinergic neurons were obviously decreased after injury, the 8^th day post BC reached the lowest level, then increased gradually. The activity of ChAT expression in the BRF declined on the first 2 days after injury, then rose gradually, the peak was at The 24^th day. (2)the declining tendency of M_iR expression was observed in CA1-4, thalamus, PFC, VDB, CPU and MSN. However, except thalamus areas at 1d,4d and 16d after injury (P<0. 05 vs control) , all test point were no significantly difference with control. (3)The M₂R expression in CA1-4 declined slightly after injury but no significance (P>0. 05). The M₂R expression in thalamus decreased significantly (P<0. 05) at ld,8d and 24d. It's interesting that the M₂R expression in PFC,VDB,CPU and MSN instantly increased after injury and then rapidly decreased but no significance(P>0. 05). furthermore, the dowen regulation of M₂R was longer then M₁R'changes after injury, (4)the expression of NR in CA1-4, thalamus, PFC, CPU and VDB slightly declined after injured(F>0. 05). Conclusions (1) the Rats' Quantitative Coma Standard is a useful and objective graded standard for classification Mild and Moderate TBI. (2) The BC rats had different types of cognitive deficits and retrograde amnesia which related to the severity of TBI. The BC injury created a reversible equilibrium function deficit and motion learn- ing disorder which also related to the severity of TBI. (3) There were some diffuse degeneration and necrosis in neurons and neuroglia cell, nerve fibers injury and small vessels impairment after BC. The secondary insult after BC appeared two histopathological change peaks in early and late phases. (4) BC can cause some loss of cholinergic neurons and the down regulation of M1, M2 and N acetylcholine receptors, which may be correlated with cognitive deficits in BC rats.