| Brain radiotherapy is an important and effective treatment method for brain tumors.However,radiation-induced brain injury is a severe complication.Cognitive function deficits are the most common and severe long-term complications after radiotherapy.With the improvement of the comprehensive treatments,the survival time of patients with malignant brain tumor is significantly prolonged,and the probability of cognitive dysfunction after radiotherapy has increased.Eliminating the delayed side effects caused by radiotherapy will significantly improve the quality of life and lighten the social burden of patients with malignant brain tumors.Therefore,it is valuable to study the pathogenesis and treatment of cognitive dysfunction after brain radiotherapy.Most of the researchers focus on clinical data analysis and epidemiological investigation for the studies of radiation-induced cognitive dysfunction.However,there are few basic experimental studies and lack of systematic animal experiments or clinical correlation studies.It is inferred from previous studies that myelin sheath injury,neurogenesis decline,vascular injury,inflammation and so on may lead to radiation-induced brain injury.The definite mechanism of cognitive dysfunction caused by radiotherapy is still unclear.In the central nervous system,calcium channels are very important.They can mediate the normal function of neurons and participate in many physiological and pathological signal transductions after activation.Neurodegenerative changes caused by acute central nervous system injury have been reported to be related to apoptosis and oxidative stress induced by calcium influx.In the study of cerebral ischemia,calcium channel blocker nimodipine can protect brain tissue and reduce neuron apoptosis.Calcium channel antagonists can not only selectively dilate cerebral vessels,but also improve memory and promote the recovery of neural function in brain injury and cerebral ischemia.We expect whether calcium channel disorder plays a key role in cognitive dysfunction after radiotherapy and whether calcium antagonists can improve and prevent cognitive dysfunction caused by radiation brain injury.The purpose of this study is to investigate the cognitive impairment associated with radiotherapy in elderly patients with glioma after radiotherapy,and to establish a rat whole brain radiotherapy model.Then we plan to discover the pathogenesis of cognitive dysfunction induced by brain radiotherapy and to explore the feasibility of calcium antagonist nimodipine in the treatment and prevention.Part 1 The clinical study for cognitive dysfunction after radiotherapy inthe elderly glioma patientsObjective:The aim of our study is to analyze the clinical features and treatment options of gliomas in the elderly,evaluate the prognostic factors and observe the survival time and cognitive dysfunction of the elderly glioma patients who received radiotherapy after operation.The study will provide preliminary clinical data for further research.Method:Patients’information was extracted retrospectively from the database of the Department of Neurosurgery in the Fourth Hospital of Hebei Medical University between Jan 2009 and Dec 2012.Fourty-three elderly glioma patients who were over 60 years old from the total 197 glioma patients were identified by surgeries in this study.Fourty elderly patients finished the follow-up.The shortest follow-up period was over three years.The histology was proved by craniotomy or biopsy.After operation,radiotherapy alone or radiotherapy with chemotherapy,chemotherapy alone or conservative treatments were performed.The patients had no previous history of mental illness and had normal understanding and communicating skills.The data included sex,age,tumor location,preoperative KPS score,pathology,treatment including surgical resection,postoperative radiotherapy and dose,chemotherapy,cognitive function,as well as survival time.Mini-mental State Examination(MMSE)was used to evaluate the cognitive function.The score of 27 to 30 was considered as normal cognitive function,and the score<27was considered as cognitive dysfunction.All analyses were conducted with SPSS20.0 software package.The survival data was used Cox regression analysis including Univariate and Multivariate analysis.P values less than0.05 were considered statistically significant in Univariate analysis.If0.05<P<0.2,Multivariate analysis was further performed.When the followed P value was less than 0.05,the difference was significant.Results:Adjuvant radiation after brain tumor surgeries can prolong the survival time in elderly gliomas.From the data observed in our study,the mean survival time of patients with no radiation after brain surgeries was6.17±5.25 months.However,the patients who got unfinished radiation had a mean survival of 21.92±4.06 months.For the standard radiation group,the mean survival time was long to 32.08±22.5 months.By univariate analysis for with radiation and no radiation therapy,compared with radiation group,the RR(95%CI)of one year survival in no radiation group was 20.52(4.46-94.41)(P<0.001)and RR of three year survival was 16.68(3.89-71.60)(P<0.001).When followed up to May 2014,15 people(37.5%)had died of illness,25(62.5%)survived,and all the surviving patients had received postoperative radiotherapy.12(48%)had normal memory and understanding ability,13(52.0%)had different degree of memory loss or misunderstanding and their MMSE scores were less than 27.Of the patients with cognitive impairment,61.5%were accompanied with systemic diseases(hypertension,diabetes).By December 2015,only 8 survived,of which 5(62.5%)had cognitive impairment in varying degrees which their MMSE scores were less than 27.Summary:Radiation therapy after brain tumor surgeries can prolong the survival time for elderly gliomas but it can also aggravate cognitive fuction deficits.Over half patients had varying degrees of cognitive problems after radiation when the mean following up time was close to two years.And cognitive problems happened more in the elderly gliomas with systematic diseases.Part 2 The establishment of a rat whole-brain radiotherapy model and the feasibility of post-radiotherapy calcium antagonist in the treatment of cognitive dysfunction after radiotherapyObjective:To establish a single-time whole-brain radiotherapy model in rats,and to establish a simple and effective animal model for cognitive impairment after radiotherapy.It is also to explore the possibility of calcium antagonist in the treatment of cognitive dysfunction after radiotherapy.Methods:Sixty ten-week-old healthy male Wistar rats were randomly divided into three groups which included the whole-brain radiotherapy group(WBRT and saline-injection),Control group(injection of saline)and the nimodipine group(WBRT and nimodipine treatment).Each group had 20 rats.The endpoint time was at 7 days(10 rats in each group)or 12 weeks(10 rats in each group)post radiation.The WBRT group was given a single whole brain irradiation with a dose of 30 Gy,dose rate of 300 cGy/min and a source scale of 100 cm.In Control group,only anesthesia was received,and the nimodipine group was injected with 2.5 ml/kg nimodipine injection(0.5 mg/kg)after radiation once a day for 7 consecutive days.The rats were fed to 12weeks,and the rats were sacrificed before tested by water maze test to test the cognitive function.Results:All the rats were alive during the whole experiment,and no death occurred during the course of the anesthesia and the process of radiation.After 3 days of brain irradiation,the weight of rats in the WBRT group was significantly less than that in Control group(P<0.05),but there was no significant difference in the weight of WBRT group and Control group at the end of the first month.In the irradiated area of the rat,we could observe that the hair fell off,the head skin was damaged.The rats had low activity and poor appetites short-termly.Then they turned to over excited,restless,aggressive and the mutual tear.Cognitive function of the rats was tested by Morris water maze test.The latency of the platform was 28.80±18.13s(Control group),35.76±20.94s(WBRT group)in the first day of training.The latency of nimodipine group was 30.48±17.83s.Compared with WBRT group,the latency of nimodipine group was shortened,P=0.019.The results suggested that learning ability declined after the whole-brain irradiation,and the application of nimodipine could improve learning ability.In the space exploration test,ratio of WBRT group in quadrant of the platform(24.33%±1.17%)was significantly lower than that in Control group(29.63%±2.25%),P=0.001.In the nimodipine group,the ratio of the platform quadrant distance/swimming distance(28.24±2.36%)was higher than that of WBRT group.The results showed that cognitive function of WBRT group was declined compared with that of the control.Nissl staining showed that the number of normal neurons in the hippocampal CA1 region of WBRT group decreased,and that of nimodipine group was improved than WBRT group,but still lower than the control.Conclusion:The decrease of the number of neurons in the hippocampus in rats after WBRT indicates that the function of the neurons after brain radiation is damaged to a certain extent.The Morris water maze test demonstrated that the learning and memory capacity of the rats decreased after brain irradiation.After radiotherapy followed by nimodipine injection,the learning and memory function of the rats improved,and the number of neurons in the hippocampal CA1 region of the nimodipine group was increased than that of WBRT group.It can be preliminarily determined that a simple and effective animal experimental model of cognitive dysfunction after radiotherapy can be established when a single dose of 30-Gy whole-brain radiotherapy is given to a rat.It is feasible and effective to use the calcium antagonist nimodipine for the treatment of radiation induced cognitive dysfunction.Part 3 Effects of whole brain radiotherapy and calcium antagonists onneurons of rat hippocampusObjective:To explore the changes in morphology and number of CA1neurons,expression of Caspase3 in dentate gyrus of rat hippocampus after whole brain radiotherapy and calcium antagonists’treatment.Methods:Three months after WBRT,rats were anesthetized with chloral hydrate and perfused with normal saline followed by 4%paraformaldehyde(0-4℃)through ascending aorta and brain tissue was quickly dissected out.Brain tissue 1~4mm behind chiasma opticum with coronal incision were fixed at 4%paraformaldehyde for 24 hours,followed by gradient alcohol dehydration,paraffin embedding,slice thickness of 5 um.After dewaxing,histological staining was carried out for 5 discontinuous slices from each rat.After Nissl staining,the number of pyramidal cells with intact membrane,full nucleus and clear nucleolus in the 1mm of the bilateral hippocampal CA1region was counted with light microscope.We counted two areas for each side of the hippocampal CA1 area and average the neuron number.The number of surviving neurons was calculated according to the neuron number/mm~2 in hippocampal CA1 region.In addition,5 discontinuous paraffin sections were taken from each rat for Caspase-3 immunohistochemistry staining to detect the apoptosis of neurons in the dentate gyrus of the hippocampus.Results:Histological staining showed that the number of neurons in the hippocampal CA1 region(85.3±22.9/mm~2)was significantly decreased than that in Control group(183.2±27.6/mm~2)after whole brain irradiation.Some of the neuron cells were shriveled,necrotic and vacuolated.The expression level of caspase3 antibody in hippocampal dentate gyrus(DG)neurons was increased.Compared with WBRT group,the number of neurons in the hippocampus(128±19.5/mm~2)of the nimodipine group increased and the expression of caspase3 antibody in the DG area decreased.However,compared with the control group,the number of neurons in the hippocampal area in the nimodipine group was still significantly decreased than that in the Control group and the expression of caspase3 antibody was increased.Summary:The apoptosis of hippocampal neurons increases after radiotherapy,which leads to a decline of neuron number,further affects the learning and memory function,and leads to cognitive dysfunction after radiotherapy.The calcium antagonist nimodipine could partly down-regulate the expression of caspase3 in dentate gyrus neurons,inhibit the apoptosis of hippocampal neurons,and thus improve the impairment of learning and memory function after whole brain radiotherapy.Part 4 Effects of whole brain radiotherapy and calcium antagonists onneural regeneration and apoptosis related gene expression in hippocampusObjective:To investigate the expression of genes related to cognitive dysfunction after whole brain radiotherapy and whether calcium antagonists can affect the expression of these genes.Methods:7 days or 3 months after the whole brain irradiation,the rats were anesthetized with chloral hydrate,and then the brain was dissected out quickly and placed on ice.The hippocampi were isolated from both sides of brain hemispheres.After stored with liquid nitrogen and extracting RNA according to procedure,the expression levels of Bax-2,Bcl-2 were detected by RT-PCR in 7 day group rats and Bax/Bcl-2 ratio was calculated.APP,BDNF,GAP-43 were detected by RT-PCR in 3 months group rats.We then could observe some early and delayed changes of hippocampus after whole brain irradiation and nimodipine treatment in rats.Summary:After WBRT,the Bax/Bcl-2 ratio in the hippocampus is up-regulated,suggesting that the apoptosis of neurons is increased.After nimodipine,the Bax/Bcl-2 ratio in hippocampus was significantly lower than that in WBRT group,suggesting that the apoptosis of neurons in hippocampus after radiotherapy decreased by nimodipine treatment.3 months after WBRT,the levels of BDNF,GAP-43 in hippocampus down-regulated and APP was up-regulated in hippocampus.However,nimodipine could up-regulate the expression of BDNF,but did not change the expression of GAP-43,APP after radiotherapy.In conclusion,the mechanism by which nimodipine inhibits the apoptosis of hippocampal neurons after radiotherapy may be related to its inhibition of the up-regulation of Bax/Bcl-2,thereby protecting the expression of BDNF in the hippocampus.Conclusions:1.For elderly glioma patients,postoperative radiotherapy can prolong survival time,and cognitive dysfunction may occur after radiotherapy.If accompanied by hypertension,diabetes,the elderly glioma patients with postoperative radiotherapy are more likely to develop cognitive dysfunction.2.A simple and effective animal model of cognitive dysfunction after radiotherapy could be established by 30 Gy single dose of whole brain radiotherapy in rats.3.After whole brain irradiation,the number of neurons in the hippo-campal CA1 region decreased and the expression of caspase3 antibody in the dentate gyrus neurons increased significantly.Nimodipine could inhibit the apoptosis of neurons in the hippocampus after radiotherapy and improve the impairment of cognitive function after the whole brain irradiation.4.The mechanism by which nimodipine inhibits apoptosis of hippoca-mpal neurons after radiotherapy may be related to its inhibition of up-regulation of Bax/Bcl-2,which protects the expression of BDNF. |
PDF Full Text Request