| Inflicted traumatic brain injury is a common central nervous system disease,is most between 3 and 6 months of age and is associated with a high mortality rate(10-40%).The most recent report from the US Department of Health and Human Services indicates that inflicted traumatic brain injury is the leading type of intentional injury in infants and young children and ranks the third leading cause of serious head injury for children 0 to 14 years old. Acute ophthalmologic and neurological signs, and poor neurological outcomes happen in more than half the cases Early appearing neurological problems include cerebral palsy and mental retardation, blindness, and epilepsy, while long-term follow-up studies have shown microcephaly associated with cognitive and major behavioral problems.A prospective study from Scotland with a mean follow-up period of 59 months showed 68% of chil,dren with AHT have abnormal developmental findings in follow-up, and 36% developed severe cognitive and behavioral difficulties, making them totally dependent on parents and caregivers.The economic impact of i TBI is immense for society and families, often costing 2 to 3 times more than TBI from other causes. The direct cost of care for child maltreatment in the United States was greater than US $24 billion in 2003. Reported incidence rates are grossly underestimated because of challenges in establishing the diagnosis of abuse.Therefore, animal models of i TBI(inflicted traumatic brain injury), especially for rotational-acceleration traumatic brain injury(TBI), are essential for testing novel hypotheses, pathological mechanisms and therapeutic interventions. Unfortunately the modeling of this unique TBI was not well developed. In this study, we introduced a new mouse model to generate rotational-acceleration brain injury.Part 1 Establish a new inflicted traumatic brain injury modelPurpose:(1)Developing a inflicted traumatic brain injury modelMethods:(1)Animal model establishment: Developing a new rotational-acceleration traumatic brain injurymodel that can mimic the injuries to infant observed clinically.The eight postnatal day12(P12) C57/BL6 mouse pups wereculled by body weight from each litter and divided into two groups for exposure to either inflicted insult or sham treatment as control. P12 pup was placed into an anesthesia box for 3 minutes that filled with 3% isoflurane that vaporized by 100% oxygen. Each animal lied on a horizontal plate in a prone position and its head will be firmly fixed by a rubber band in a head holder. The head holder will be rotated when hit by a piston. The velocity of rotational acceleration and angle was adjusted by pneumatic pressure and moving distance of piston. One time rotational acceleration procedure was profiled as 60 psi for 60 times with flexion +35°,extention-25° rotational angle. After the procedure the mortality was counted.(2)Evaluation of consciousness: The righting reflex, also known as the Labyrinthine righting reflex, is a reflex that corrects the orientation of the body when it is taken out of its normal upright position. It is initiated by the vestibular system, which detects that the body is not erect and causes the head to move back into position as the rest of the body follows,the reflex center is in the mesencephalon. When the trunkis stillinanirregularposition,neckmuscles distortionoccurs,andthe stimulationitself rendered the second reflection that maketrunk to the normal position.Rightingreflexisused to evaluatethe time which recovered from anesthetic status.Results:(1)compared with control group,under 60 psi for 60 、 80 or 100 times rotational-acceleration procedures, apnea, cyanosis appear in all i TBI group members which means hypoxia occur.Under the condition of 100 time insults the survival rate in i TBI group is 79%.(2)Righting reflex result shows:the average time of 83 seconds in control group that recovered from anesthetic status to consciousness.,under 60 psi for 60、80 or 100 times rotational-acceleration procedures,the average time of consciousness regain specifically is, 181 seconds, 266 seconds, 328 seconds. increased gradiently.Conclusion:This study successfully established a rotational-acceleration i TBI model in developing mice.Part 2 Neuropathology and physiology alterations after i TBI in developing micePurpose: To study the neuropathology and physiology alterations after i TBI in developing mice.Methods:(1) the vital signs detection:the mice was subjected in prone position with a sensor clip on thigh root after the rotational-acceleration insults. cardiopulmonary function, and oxygen saturation were monitored by the Mouse Ox TM Oximeter(STARR Life Sci. Corp.). Analog data were continuously digitized by a computer interfaced with Win Daq data acquisition system.(2) brain damage assessment: parietal removal after the injury, make a observation of whether Subdural/subarachnoid hemorrhage or edema occurs; To test the blood brain barrier permeability by Evans Blue absorption; To test the brain edema after injury by measuring the wet/dry weight of the brain.Results:(1) After exposed to rotational-acceleration head insults for 60 times at the severity of 60 psi, all the pups showed irregular respiration and some had the central apnea. Compared to sham pups, Their respiratory rate, heart beating rate, and tissue oxygenation were significantly affected.(2)under different conditions of insult(15psi,30times; 30 psi,30times;60psi,30times; 60 psi,30times; 60 psi,60times; 60 psi, 100times), Subdural/subarachnoid hemorrhage can be found in i TBI group.7 hours post injury with 60 psi for 60 times insult,compared with control group, the amount of EB in tissue extracts was measured by absorbance at 610 nm increased in i TBI group. There are significant differences(P<0.05); 7 hours post injury with 60 psi for 60 times insult, wet/dry ratio of the control group is4.74±0.15 g / g, the wet/dry ratio of the i TBI group5.05±0.24 g / g,difference is statistically significant(P<0.05). Conclusion: i TBI elicits a transient cardiopulmonary dysfunction and the changes of vital signs, including bradycardia,apnea and gradually returned to normal breath-rate, blood oxygen saturation gradually goes to normal levels; Blood-brain barrier disintegribility,, increase of capillary permeability and edema in brain parenchyma.Part 3 Behavioral changes after i TBI in developing micePurpose:To study whether the i TBI can change the locomotoive and cognitive function in developing mice.Methods:(1)Behavioral tests: Rotarod test was used respectively in different time points after injury(9,19, 30 days after the injury) to evaluate neural function, and observe the recovery of locomotive function and coordination in male mice. Ymaze test was used respectively in different time points after injury(14, 21, 28 days after the injury) to evaluate neural function,to observe the recovery of distinctiveness of learning and memory ability.(2)Expression of molecule:collect fresh brains from control group/i TBI group 3days post injury(60psi,60times),quantitatively analyze the glial fibrillary acidic protein(GFAP) and interleukin 6(IL-6) 、 Ibal-1 expression,to observe proinflammatory and astroglial responses influence on early behavioral test.(3)Neuronal injury assessment:collect brains 30 days after injury(60psi,60times)and fixed in 4% paraformaldehyde, dehydration by 20% sucrose, embedding on dry ice.Then sectioning on cryostat and soaked in 4% paraformaldehyde over 7 days for silver stainging.To evaluate how the neuronal injury influence the long-term behavioral test at morphological level.Results:(1)Rotarod test results in two groups show that: the score value in two groups arise gradually from 9-30 days post injury. 9 days post injury control /i TBI=81.37s/50.16, difference is statistically significant(P<0.05),19 days post injury control/i TBI=109.12s/129.2s, difference is not statistically significant. 30 days post injury control/i TBI=120.36s/148.96 s, difference is not statistically significant.Ymaze test results show that: 14-28 days post injury,the score value in control group has no significant difference,but have a apparently decrease at 28 days after injury in i TBI group,14 days post injury control/i TBI=0.59/0.58, 21 days post injury control/i TBI=0.56/0.41,28 days post injury control/i TBI=0.59/0.58, difference is statistically significant(P<0.05).(2)Western blot result shows that: 3 days post injury, glial fibrillary acidic protein(GFAP) and interleukin 6(IL- 6) in levels have increased significantly i TBI group and compare with the control group,the difference is statistically significant(P<0.05)(3)silver staining results show that: 42 days post injury, black grains have been found in primary motor cortex(M1),primary somatosensory cortex of forelimb region(S1FL),olfactory tubercle(OF; in basal forebrain(BF),indicate undergoing degeneration in these areas.Conclusion:Inflicted traumatic brain injury has a great impact on locomotive function and coordination in adolescent stage,but return to the nomal levers in the late development.However, the impact on the cognitive function appears inyoung adult stage. To sum up, the results show that neonatal i TBI results in locomotive dysfunction, coordinative and cognitive deficits in developing mice,induces pro-inflammatory/astroglial responses at early development and neuronal degeneration in late development, it may also be the main factor that lead to behavioral changes in function. |
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