Circadian Disruption Worsens The Pathological Changes And Inhibits The Treatment Effect Of Nerve Growth Factor In Adult Rats After Traumatic Brain Injury

Research BackgroundTraumatic brain injury(traumatic brain injury, TBI) is very common, high mortality and morbidity, brings great burden to the society. Its pathophysiologic process can be divided into primary injury and secondary injury. Primary damage refers to violence injury was suffered from mechanical damage caused brain tissue deformation, cerebrovascular, axons and neurons and glial cells by direct damage, the damage can’t through the implementation of clinical intervention, only to strengthen prevention. Secondary damage in the primary damage immediately after beginning,local neurochemical changes cause a series of biochemical changes, cell injury and repair, proliferation and apoptosis, metabolic change interactions and cellular events in life comes to an end. The pathophysiology of secondary brain injury complicated and deep understanding of the pathological factors affecting brain injury, for the pathological understanding and treatment of TBI will bring great breakthrough.Study confirms that the biology of sleep and awakening, eating behavior, and a variety of physiological, biochemical, metabolic process follows the periodical change of about 24 hours, called a circadian rhythm(circadian rhythms).Circadian rhythm is a variety of life on the earth cycle of environment factors change the result of the long-term adaptation and evolution, it widely exists in various kinds of organic life, is life important phenomenon in the nature.Circadian gene regulation, including stem cells, a variety of cell proliferation,participated in the reconstruction of repair; In the course of evolution of biological clock control organisms adapt itself to the changing environment of circadian rhythm,better use of energy for life, so involved in energy metabolism is one of the important performance of the circadian clock to participate in the activities of life; Clock in the cell cycle process, the influence of neurotransmitter release and neural plasticity.Currently, more and more epidemiological and genetic data showed that the destruction of the circadian rhythm is closely related to the pathological state, these pathological states including depression, sleep disorders, metabolic syndrome and cancer.After brain injury patients facing environmental and pathological damage, the effects of circadian rhythm disorders, studies have shown that acute period of sleep disorder after brain injury, recovery sleep disorders. Animal experiment and clinical study Imbalance circadian gene expression studies have found that after brain injury after brain injury clock genes expression disorder, so we speculated that circadian rhythm control function of the brain, participate in pathological damage process, but how to participate in and potential of pathological mechanism is unclear; At the same time, the study found that circadian rhythms involved in cell cycle progression, affect insulin, glucocorticoid and the activity of antineoplastic drugs, in the brain injury treatment, model NGF can promote the growth of nerve cells, and differentiation, can promote restoration of the damaged cells, but under the environment of circadian rhythm disorders, influence on NGF plays the lack of experimental data, this research will study from these two aspects.Chapter I Circadian rhythm disorders affect hippocampal formation is aggravating the pathological damage of brain damageObjective:to observe the circadian rhythm disorders influence on brain injury pathological changes and the impact on the hippocampus nerve regeneration and the possible mechanismMethods:Preparation of craniocerebral injury of rats model, Use the normal Light and shade environment(Light/Dark, LD) and night continuous Light(Light/Light, LL)two environment model(i.e., the circadian rhythm disorders and normal circadian rhythm), continuous intervention in 14 days.Sixty adult male Sprague–Dawley rats were subjected to four groups:Sham/LD group、 Sham/LL group、 TBI /LD group、 TBI/LL group. Using the neural functional score observation rat behavior change; The balance beam balance experiment and walking experiment testing rats movement and balance ability; Water maze test rats learning and memory ability change;14 days take brain tissue specimens, calculating volume of focal brain injury; Nissl staining to observe the damage area of cortex and hippocampus neuron survival cell number; The Brdu mark newborn live cells, Ki-67 proliferation cells, DCX marking new immature neurons,immunohistochemical determination of hippocampal DG area 14 days students quantity, cell proliferation and cell survival number of immature neurons.Results:1. The comparison between groups of rats, water of weight and food intake,found that 14 days compared with the Sham/LD group, Sham /LL group rat body weight, food intake increased significantly(P < 0.05), water quantity without difference(P > 0.05);TBI/LD group compared with Sham/LD group, body weight and nerve function score lower(P < 0.05);TBI/LL with TBI/LD group compared to body weight, feeding and nerve function score were lower(P < 0.05).2. The balance beam experiment and water maze experiment results show that compared with the Sham/LL group, TBI/LD group balance exercise capacity is reduced, the ability of learning and memory by obvious damage(P < 0.05);TB/LL group rats with TBI/LD balance ability of rats, motor coordination and cognitive decline further(P < 0.05). 3. The Nissl staining can damage volume results show TBI/LLgroup injury is bigger than the volume of TBI/LD group(34.33 ±1.58 mm3 VS 23.97 ±2.94 mm3 P <0.05); TBI/LL group brain damage around the area and the decrease in the number of hippocampus CA1 area neurons survival, but no difference between the number of cells in the areas of hippocampal DG area.4. Brain damage after TBI/LD group compared with Sham/LD group, the hippocampal DG area Ki-67 positive cells number, Brdu tag number of newborn cells survive also increased(P < 0.05); TBI/LLgroup of hippocampal DG area Ki-67 positive cells number and Brdu tag number of newborn cells survive a Sham/LD and TBI/LD group decreased significantly(P < 0.05).5. DCX positive cells with immature neurons, TBI/LD group compared with Sham/LD group, the hippocampal DG area increase in the number of immature neurons, but TBI/LLgroup of hippocampal DG area number of immature neurons decreased significantly(P < 0.05).Conclusion:Rats after injury associated with circadian rhythm disorders will make its weight loss, neurologic symptoms aggravated, balance the sport ability and cognitive ability damage, injury rats volume increased, at the same time, the brain neuron survival number and the decrease in the number of new neurons.Chapter II the mechanism of circadian rhythm disorders affect NGF treatment after traumatic brain injuryObjective:to study the circadian rhythm disorders affect NGF on the molecular mechanismsMethods:Preparation of craniocerebral injury of rats model, Use the normal Light and shade environment(Light/Dark, LD) and night continuous Light(Light/Light, LL) two environment model(i.e., the circadian rhythm disorders and normal circadian rhythm), continuous intervention in 14 days. Sixty adult male Sprague–Dawley rats were subjected to three groups: normal control group(Sham/LD) and brain damage normal rhythm model NGF treatment group(TBI/LD/NGF), brain injury with circadian rhythm disorders model NGF treatment group(TBI/LL/NGF).Model NGF treatment by 32 ug/kg dose muscle injection, injection of 14 consecutive days. Using the neural functional score observation rat behavior change; The balance beam balance experiment and walking experiment testing rats movement and balance ability; Water maze test rats learning and memory ability change; In real time quantitative PCR rats had NGF plays Trk A and p75 receptor(NTR) pathways related gene AKT, NTRAK1, NFK beta, the BCL- 2, BAX, JNK, Caspase3 m RNA in the relative expression of transcription; Westernblot determination model NGF receptors signaling pathways Trk A, p75, JNK, Caspase3, AKT, NFK beta, NCo R, PARP protein content; Immunofluorescence observations Trk A, p75 receptor expression and star-shaped glial cell proliferation, microglia activation;TUNEL method to detect brain damage area apoptotic cells; Elisa method to detect content of inflammatory factor beta, IL- 6, IL- 1 TNF-β in serum and brain tissue.Results:1. The neurological function score of each group rats and the experiment results show that the balance beam, TBI/LL damage restore neural function in rat model NGF group was obviously delayed in TBI/LD/ NGF group, 14 days after injury of the balance beam balance time significantly shortened, walking at the same time prolonged(P < 0.05).2. The water maze experiment, positioning cruise phase, TBI/LL/NGF group with circadian rhythm disorders, lengthen the incubation period, learning ability is lower than the TBI/LD/NGF group;In space exploration stage, TBI/LD space exploration time and number of cross platform model NGF group was obviously more than the TBI/LL/ NGF group.3. Real-time quantitative PCR showed that TBI /LD/NGF plays in the circadian rhythm NGF plays normally high affinity receptor Trk A, related genes, cell growth and differentiation AKT, NTRAK1, NFK beta, heighten the BCL-2 expression, low affinity p75 and apoptosis related gene BAX, JNK, Caspase3 expression; Instead under the condition of circadian rhythm disorders Trk A, AKT, NTRAK1, NFKβ,reduce the BCL-2 expression, p75 and apoptosis related gene BAX, JNK, Caspase-3expression showed a trend of increase. At the same time Westernblot results consistent with the m RNA expression.4. Immunofluorescence found in TBI/LD/Trk A receptor positive cells model NGF group was obviously more than the TBI/LL/ NGF group(P < 0.05), but the p75(NTR) positive cells is less than the TBI/LL/ NGF group(P < 0.05).TUNEL staining showed that TBI /LD/ NGF group was obviously less than TBI/LL/ NGF group(P <0.05).5. Immunofluorescence of GFAP positive astrocytes markers showed in TBI/LL/astrocytes reactive hyperplasia model NGF group was obviously more than the TBI/LD/ NGF group(TBI/LL/NGF:466.90±65.10/field VS 339.56±48.37/ field,P<0.05);Microglia activation with circadian rhythm disorders also showed a trend of increase(TBI/LL/NGF:115.60±23.62/field VS 65.40±12.55/field,P<0.0).6. In the serum and brain tissue inflammation factor beta, IL-6, IL-1 TNF-βsecretion TBI /LL/ NGF group was obviously higher than that of TBI/LD/ NGF group(P < 0.05).Conclusion:Under the condition of circadian rhythm disorders, NGF plays for the treatment of brain injury effect is reduced, and the model NGF compared under the condition of normal rhythm, increase nerve dysfunction and cognitive impairment, increased number of apoptosis, increased inflammatory cytokines release, with circadian rhythm disorders after activation of low affinity receptor p75 mediated apoptosis(NTR) signal path expression.