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The number of research has been done on the changes of neurotropins levels in suicidal brains. And this is all because of the biochemical changes in suicidal brain.
Generally the analysis of the levels of the BDNF (brain-derived neurotrophic factor) and NT-3 (neurotrophin-3) is main biochemical neurons measured in the suicidal victim.
There is also some experiment has done on the NT-3(neurotrophin-3), BDNF (brain-derived neurotrophic factor), abnormalities of tropomycin receptor kinase (TrkB), P75NTR .
Here in this research they have examine weather there is an abnormality in the BDNF and neurotrophin-3 proteins in suicide victim and effects of antemortem diagnosis and psychotropic drugs.
Neurotropins are impotent regulatory of neuronal plasticity in the developing and adult brain.
Dwivedidy, Mondal AC, Rizavi HS, they have done the research on the changes in the expression of neurotrophins that associated with suicide brain.
Here they have measure the massager ribonucleic acid (mRNA) levels of nerve growth factor ,Neurotrophin (NT-3), NY-4/5 , cyclophilin, (1)
Neuron-specific enclose by quantitative reverse transcriptase polymerase chain reaction and about protein levels of neurotropins were determine by enzyme linked immunosorbent assay that is ELISA and prefrontal cortex (PCF)and hippocampus from 28 suicide victim and 21 control subject. So they examine the suicide brain associated with change in expression of neurotrophines.
In this topic they were mainly discuss on the suicide in modern societies. The number of suicide occurs due to the affective illness, stress, depression. And the several studies state that the biochemical changes in post-mortem brains. Some alteration has taken in serotoninergic system in the patient who commuted suicide. Still the exact pathway and the mechanism of the dysfunction has not properly describe or examine. (2)
According to the resent studies of the effect of neurotrophins on brain and brain imaging studies have been reported changes in volume of hippocampus and PCF (prefrontal cortex) in the patient suffered with depression and other bipolar disorder. Here BDNF (brain-derived neurotrophic factor) and NT-3(neurotrophin-3) were assayed by western blot or with ELISA (enzyme linked immunosorbent assay).All neurotrophins are structurally related to the NGF (nerve growth factor) which play important role in neuronal survival, differentiation and plasticity during development and adult hood. The link between the function of 5 HT (5-hydroxytryptamine) and BDNF (brain-derivedneurotrophic factor) function inversely proportional. According to the some studies state that the stress can decreases the function of BDNF. When administered to stress animals, BDNF was shown to produce antidepressant effect by antagonizing behavioural models of depression, the data of human study support the role of the BDNF and NT-3 in pathophysiology of stress and depression and in action of antidepressant drugs. By the genetic studies reported that the BDNF or its low affinity receptor, p75, gene polymorphism associated to depressive disorder. From above investigation examine the weather any abnormality in the brain BDNF and NT-3 proteins levels and this abnormality related to the depression. And group of suicide victim with either depression were identified and compared to non- psychiatric non suicide controls.
Here in this experiment the hypothesis state that a decrease of BDNF and NT-3 levels in the hippocampus and partly in the ventral PFC, but not in the entorhinal cortex.
Mainly three method are use for the quantification of neurotrophin are following :
Quantification of BDNF protein with Western Blot technique
BDNF levels were immunolabeled with the Western blot technique. coronal slices were homogenized in a lysis buffer, the content of buffer are 100mM Tris, 150 mM NaCL, 1% Triton X-100, 1% sodium decolate, 0.1 % SDS, 5 mM EDTA, 1mM phenylemethylsulfonyl- fluoride(PMSF), 10 µl/ml aprotinin, 1µl/ml leupeptin, and 1µl/ml pepstatin. That homogenates were incubated for 20 min at 40 C than shaking centrifuged and the supernatant are taken for experiment. The loding buffer contained glycerol, Mercaptoethanol, Bromophenol blue. Then the sample were heated at 950 C for 10 min before gel loading than equal amount of soluble fraction of brain protein was electrophoresed on 15% sds-poly acralamide gel with a mini protean system with molecular weight standards. Mainly gel was prepared in the lab abd that is bio-red, after that electrophoresis the sample was electro transferred on to the PDVF membranes. This electro transferred depends on the antibody sensitivity, than block it for 1 hour at 370C in TBST solution with 5% non fat milk power and bovine serum albumin 2%. Than incubated overnight at 4% with primary antibody for BDNF. Washed and incubated with anti rabbit IgG labelled with HRP for 1hour at 370C than developed membrane with chemiluminesence ELC western blottings and exposition of membrane with radio graphic films.
Quantification of BDNF and NT-3 with the ELISA method :
Coronal slices was homogenised in a lysis buffer the homogenates was incubated for 10min at 40C centrifuge, shaking and supernatant used for the experiment. This technique was performed by using BDNF or NT-3 Emax immunoassay system kit, this kits design for the detection of BDNF and NT-3. In this technique Nunc Mazsorb 96 well plats coated with monoclonal antibodies that is anti-BDNF and anit- NT3 diluted with the carbonating coatings buffers incubated at 40 over night. After that empty the coating before blocking solution was added for 1 hour, wash it 5 times before adding the sample. Than add specific polyclonal antibody than incubated at 370C , wash 5 times before further incubation with the IgY antibody conjugate to 2hour for BDNF or may be 2 hour 30 min for NT-3 than with peroxidise substrate that is TMB (tetra methylbenzene) for 1 hour to gives color than sample was acidify with acetic acid. And for the determination of BDNF and NT-3 levels in homogenates microplate reader sat at 450 nm. Here intra assay and inter assay variation were Ë‚5% and 8% respectively.
Advantages and disadvantages of ELISA test:
Advantages are the ELISA tests are widely used to detect the substance that has antigenic property, primarily proteins. These test are generally relatively accurate test and they are consider highly sensitive and specific and coper favourably with other method use to detect substance in the body such as radioimmunoassay test are mainly for the test no needing radio isotopes.(3)
Disadvantages of ELISA are this technique required skilled laboratory technician and specialise equipments.
Statistical Method :
The data arrived was run with the statistical software. The ANOVA is used in this experiment because the variation i in the group data and the two treatments are given to the groups. Mainly here the some group that has treated with anti depressant drugs and some are not. ANCOVA was used with PMD and age as covariates. Bonferroni test was used for multiple comparisons and signification was accepted at PË‚0.01. And variation 2 way ANOVA was used. Then the mode of suicide on BDNF/NT-3 levels was tested by comparing the violent and non violent mode of death and unpaired t test was used and significance set up at PË‚0.05.
Material and method used :
Postmortem brain samples:
For the experiment Human brains were collected at autopsy from the Institute of Forensic Medicine, Geneva, Switzerland. And this has been approved by the Research and ethic review board of the department of Psychiatry and the faculty of Medicine in Geneva. This all is done under legal procedure.
Than the brain was removed for the experiment to determine the neuropathologic abnormalities after three of brain was isolated from the from right hemisphere. Than isolate brain part was stored at -800 C.
The isolated parts are ventral prefrontal cortex (PFC), hippocampus area and the entorhinal cortex.
The suicide victims consisted of a well-defined population of thirty subjects (19 M/11 F) carefully determined by forensic physicians. The mean age was 47 F 17 years and the post-mortem delay (PMD) from death to storage was 24 F 19 h.The causes of death were hanging (n = 8), drowning (n = gunshot) wound (GSW: n = 4), drug overdose (n = 6), jump (n = 4), asphyxia (n =2), carbon monoxide poisoning (n = 2), and multiple trauma (n = 3). Retrospective searching for antemortem clinical diagnosis (with the DSM-IV) and drug treatment through reviews of medical histories of psychiatric units.
The control group consisted of twenty-four non-suicide subjects (14 M/10 F) who died from determined cause. The mean age was 45 F 14 years and the postmortem delay (PMD) was 28 F 21 h. The causes of death varied and included myocardial infarction (n = 5), pulmonary embolism (n = 2), homicide (n = 2), road traffic accident (n = 7), various somatic diseases (n =7), or accidental drowning (n = 1).
From the figure 1. That displays the western blot of immunolabeling of BDNF and β-actin band for one subject of each group. Here the BDNF identity was validated by which suppress the 14kDa but not the 46kDa. And the increasing levels of protein determined by linear range in displayed.
From the figure 2.gives mean value of BDNF protein in soluble fraction in hippocampus, prefrontal, and entorhinal cortex from non suicidal control and 3 groups of suicide victim : Drugs -free suicide victim either with depression that is group 1 or with out depression but with various psychiatric disease that is group 2. And drugs treated suicide victims with depression. The all value express the ratio of optical density on BDNF and β-actin proteins. The statistical differences were analyzed with ANOVA: In hippocampus: F3,50 = 6.15, P Ë‚0.001. Bonferroni for multiple comparions: significance was accepted at 0.01: with respect to controls: drug-free major depression group: P Ë‚ 0.001, drug-free with other psychiatric diseases: P Ë‚ 0.001; drug-treated major depressed group: P Ë‚ 0.03; NS. Bonferroni for multiple comparisons: significance was accepted at P Ë‚ 0.01: drug-free major depression group: P Ë‚ 0.001; drug-free with other psychiatric diseases: P Ë‚ 0.001; drug-treated depressed group: PË‚0.06; NS; In entorhinal cortex: F3,50 = 0.66, NS.
Here the table 2 mainly specified the measurement of the BDNF levels in post mortem brain and non suicide control subject that is essayed with ELISA method. And all statistical difference was analysed by ANOVA , Bonferroin for multiple comparison with respect to controls are drug-free MD group that is PË‚0.003, drug-free with other psychiatric diseases that is P Ë‚ 0.004, drug-treated major depressed group: P = 0.56; NS. In PFC: F3,26 = 6.9, P Ë‚ 0.002. Bonferroni for multiple comparisons with respect to control: drugs free major depression that is P Ë‚ 0.002.
And about table 3 that gives the NT-3 level in post-mortem brain area of suicide and non suicide control subject. Here statistical difference analysed with ANOVA and Bonferroni for multiple comparison yield no significant difference between groups.
Discussion and Comment:
In this experiment the study found that there is a decrease in BDNF levels in Hippocampus and ventral cortex but not in Entorhinal cortex of suicide victim compare with non-suicidal victim and also there is a decrease in NT-3 level in drugs free hippocampus but not in drug free PFC (ventral prefrontal cortex) .
Here the study on neurotrophines and the suicide still very few and this is the first study of NT-3 levels in suicide victim, so their may be a discrimination between the effect of diagnosis and drug on neurotrophines in brain level of suicide victim.
Here they have not mention a correlation between the BDNF/NT-3 levels and also variables like age and PMD.
Here the mood state of patient at the time of suicide is not known.
They have not mention the duration of the treatment of drugs.
The absence of changes might be attributing to the suitable complexity of its anatomy which makes difficulty in the dissection of specific area. (4)
There are also some studies reported on the positive correlation between age serum BDNF levels in healthy volunteers. (5)
Conclusion of the experiment observed there is a decrease of the BDNF and NT-3 in the hippocampus and up to some extant from PFC (ventral prefrontal cortex) but not the entorhinal. From this experiment they have found that there is a decrease in neurotrophines levels in suicide victim . and all this study support role of neurotrophines in the pathophysiology of suicidal behaviour and also support to the neurotrophines hypothesis.