BDNF and HSP 70 level in csf severe TBI related to cognitive function

ABSTRACT

The changes of BDNF and HSP70 level in cerebrospinal fluid ventricle of severe traumatic brain injury patients related to cognitive function

Fenny L. Yudiarto, Umar Kasan, Judajana, Jan Sudir Purba

Backgrounds:

Mechanical impact and hypoxic ischemic after traumatic brain injury (TBI) can cause some biomolecular and biochemistry changes. Clinically this can be seen as motor weakness, cognitive problems and mental disability.

The neurotrophins (NTs) are growth factors that play critical role in the development, maintenance, survival, and regeneration of the nervous system. Brain Derived Neurotrophic Factor (BDNF) is one of the neurotrophin family plays a pivotal function in synaptic and learning plasticity. Ischemia can induce Heat Shock Protein 70 (HSP70) expression that has molecular chaperone function and anti apoptotic function.

Objectives:

The aim of this study is to identify the expression of BDNF and HSP 70 from csf of severe TBI patients at day I-IV, and to know whether the changes of level of those proteins have some implications on cognition outcome or not.

Materials and Methods:

Design of this research is observational analytic using time series study. Samples from cerebrospinal fluid were taken from ventricle of 19 severe TBI patients with Glasgow Coma Scale (GCS) 3-8 (> 14 - <>

We also evaluated the cognitive function with some neuropsychological assessments such as; Mini Mental State Examination (MMSE) for screening of cognitive impairment, Trail Making Test A (TMT-A) to evaluate attention, concentration, visual tracking, and visuomotor (measured on time and errors), TMT-B to evaluate working memory (measured on time and errors), Continuous Performance Test (CPT) and Vigilance Complex Task (VCT) to evaluate attention and concentration, Digit Span (DS) forward to assess the verbal memory, DS backward to assess executive function, and Word Fluency to evaluate the language ability, and recall memory. The first neuropsychological assessments were done on the day when patient can verbal commands, and the second evaluations were 3 months from the first assessments.

Results:

The results showed that all samples distributed in normal curve, there were no significant difference between total BDNF male and female (P=0.924). There was a negative correlation on Pearson between the changes of level HSP70 day II-III and TMT-B (time) (p=0.045), mean the higher changes of the level of HSP70 day II-III patients would finish the test faster. The changes of BDNF level on day I till IV gave negative significant correlation with the TMT-A (error), mean the higher level of the changes of BDNF day I-III(p=0.002), I-IV(p=0.040), II-III(p=0.020), II-IV(p=0.014) gave less errors on TMT-A.

Conclusion:

The changes of level BDNF on day I-III, II-III, I-IV, II-IV, and the changes of HSP70 level on day II-III gave good results on cognitions including attention, concentration, visuomotor speed, visual tracking ability, and working memory. TMT is suitable test to evaluate the cognition of TBI.

Keywords: severe traumatic brain injury, HSP70, BDNF, Cognitive function

Introduction:

Some biomolecular and biochemical pathologic changes on traumatic brain injury (TBI) can cause damage of the cells, which can result in necrosis and apoptosis. Clinically, these can be seen as prolonged disability on both motor functions and cognition. The cognitive sequel will more severe especially on severe TBI, affecting among other things attention, concentration and memory.

The neurotrophins (NTs) are growth factors that play a critical role in the development, maintenance, survival, and death of the nervous system. Brain Derived Neurotrophic factor (BDNF) is one of the neurotrophin family and plays a pivotal function in synaptic and learning plasticity. BDNF activates two different receptors, the Trk family of receptor tyrosine kinases and p75NTR, a member of the TNF receptor superfamily. BDNF can be secreted by neuron and glial cells and is distributed in all brain tissues but in different concentrations. The hippocampus has the highest concentration of BDNF.

Heat Shock Protein 70 (HSP70) is a stress protein that can be produced by neurons and glial cells on stress conditions. It has molecular chaperone and anti apoptotic functions. On ischemic conditions, both BDNF and HSP70 can be expressed in high concentrations.

The aim of this study is to get the profile of BDNF and HSP70 concentration on the first day until the fourth day of severe TBI, and to know whether those profiles have any implications on cognitive outcome. Neuropsychological assessment was done by the researcher and psychologist.

Design of this research is observational analytic using time series study. Results were analyzed by same subject ANOVA, paired t test and Pearson correlation with probability value (p)= 0.05. Samples (cerebrospinal fluid) were taken from ventricle via ICP monitoring of 19 severe TBI patients (13 alive, 6 dead) with Glasgow Coma Scale (GCS) 3-8 (> 14 - < style=""> We measured the BDNF protein with double antibody sandwich anti-immunoglobulin ELISA (Indirect Sandwich ELISA with BDNF reagent produced by Promega) and HSP70 using double antibody sandwich ELISA (reagent of HSP70 made from Stressgen Bioreagent Corp catalog no. EKS-700).

We also evaluated the cognitive function with some neuropsychological assessments such as; Mini Mental State Examination (MMSE) for screening of cognitive impairment, Trail Making Test A (TMT-A) to evaluate attention, concentration, visuotracking, and visuomotor, TMT-B to evaluate working memory, strategic and planning, Continuous Performance Test (CPT) and Vigilance Complex Task (VCT) to evaluate attention and concentration, Digit Span (DS) forward to assess the verbal memory, attention and concentration, DS backward to assess executive function, and Word Fluency to evaluate the language ability, and recall memory. The first neuropsychological assessments were done on the day when patient can communicate (verbal command), on gaining consciousness and the second evaluations were done 3 months from the first assessments.

The results showed:

· All samples distributed in normal curve with sphericity test from Mauchly and with adjustment factor from Huynh-Feldt found that all samples were homogenous, except total BDNF levels on surviving patients on day II were lower than day I (P_1-tailed = 0.009), with day III higher than day II (P_1-tailed = 0.004), day IV higher than day II (P_1-tailed = 0.028) and day IV lower than day III (P_1-tailed = 0.000).

· There were no significant differences between total BDNF in men and women (P=0.924).

· These are the profiles of HSP70 and BDNF

• Evaluation of neuropsychological findings showed significant differences on MMSE (P_1-tailed = 0.023), TMT-A (time) (P_1-tailed = 0.021), TMT-B (time) (P_1-tailed = 0.019), TMT-B (error) (P_1-tailed = 0.031), VCT (P_1-tailed = 0.038), CPT (P_1-tailed = 0.029) and word fluency (P_1-tailed = 0.001) between 1^st and 2^nd assessments in severe TBI patients. Patients do better on 2^nd assessment than the 1^st evaluation.
• There was a negative significant correlation on Pearson between the changes of level HSP 70 day II-III and TMT-B (time) (p=0.045). Those results concluded that higher changes of the level of HSP70 day II-III will give a good outcome on cognition including attention, concentration and working memory.
• The correlation between the changes of BDNF level day I-IV and the cognitive evaluation, showed that the changes of BDNF level day I-III, II-III, I-IV, and II-IV gave negative significant correlation with the TMTA (error)_, meaning the higher level of the changes of BDNF day I-III (p=0.002), I-IV (p=0.040), II-III (p=0.020), II-IV (p=0.014) gave less error on TMT-A.
• Correlation between BDNF and HSP70 levels day by day with the neuropsychological evaluation, showed no improvement on cognitive outcome.

The level of HSP70 on surviving patients day III (19.76 ng/ml) was low, but total BDNF on day III was the highest compared with the other days (25.64ng/ml). There was a study showing that the BDNF exogenous with the ras/MAPK signaling can suppress the expression of HSF gene of HSP27. HSP27 also has an anti apoptotic function as inhibition of protein aggregation. This findings need further research to know whether the signaling BDNF endogenous with MAPK can also suppress the HSF gene of HSP70 so HSP70 expression was low.

The profile showed the highest concentration of BDNF was on the 3^rd day both on surviving and dying patients. Studies on animal models showed that the peak of glutamate released was also on the 3^rd day, and activation of NMDA receptor of glutamate can induce the release of BDNF. Astrocytes can also release the BDNF protein on the 3^rd day on ischemic conditions.

Mattson (2000) and Hellmich (2005) mentioned that the increase of HSP70 on the 1^st day followed by the increase of BDNF was the important factor determining whether the subjects can survive. On this study, patients who survived (see the profile on green line) the level of BDNF and HSP70 on the 1^st day had higher levels compared with levels of patients who died.

The expression of HSP70 on day 4 of dying patients was higher than the ones that survived. Ran (2004) said that over expression of HSP70 could not give protection effect but would induce apoptosis by binding with IKKg that can inhibit the release of NF-kB. The protective function of HSP70 depended on the degree and the cause of the damage.

On this study, the researcher used many neuropsychological tests because cognitive function is a complex higher function of the brain, which includes attention, concentration, visuospatial and memory.

The changes of HSP70 level day II-III gave good outcome in cognition. This was due to the expression of HSP70 on the 1^st day that could give the protection effect not only as a molecular chaperone also as an anti apoptotic. But if the stresses are very severe the HSP70 could not compensate for the huge damage to the cells.

The changes of BDNF level day I-III, I-IV, II-III and II-IV, can give good outcome on cognition. But the correlation day by day of BDNF gave the reverse. These findings can be explained that BDNF on this study was measured by ELISA which can not differentiate between proBDNF (immature type) and mature BDNF.

Beattie(2002) and Harrington (2004) mentioned that proBDNF would be released in big amounts on spinal cord and brain injury. ProBDNF has high affinity to p75 receptor that will induce apoptosis of the cells; this is opposite to the survival effect of mature BDNF. Lu (2005) mentioned the ‘yin and yang effect’ of this phenomenon of BDNF.

ProBDNF can have extracellular cleavage to the mature one by plasmin and matrix metallo protein 7 (MMP7). The findings on this study could be explained that at the beginning proBDNF might have a dominant role but from day I until day IV there would be a shift to the function of mature BDNF as a survival agent, after the release of plasmin/MMP7

On this study, the researcher had not compared cognition and the location of the injury showed by CT scan. This was because the previous study from Hellmich et al (2005), where he used fluid percussion injury on mice, showed that the pro apoptotic gene could be found on both ipsilateral and contralateral sides of the lesion and also on CA1, and CA3 of the hippocampus. Gusev (2003) mentioned that the hippocampus is a part of the brain which has a lot of NMDA receptors, so can be easily damaged by glutamate toxicity.

This research is a preliminary study on human brain to make the connection between biomolecular changes with the clinical outcome on cognition. Severe TBI has a multifactorial response like immunological, endocrinological and neurotransmitter interventions, and all of these are interconnected in the body. So the prediction of outcome could not be based on day by day evaluation but what was important was the fluctuation of the levels of BNDF and HSP70 on the csf in the ventricles.

Conclusion: The fluctuation of BDNF levels on day I-III, I-IV, II-III and II-IV gave good correlation on attention and concentration, and the HSP70 level changes in day II-III gave significant correlation on attention, concentration, visuomotor speed and visual tracking ability, and working memory. Trail Making Test (A and B) could be used as a test to evaluate the cognitive function of TBI. Better intellectual functions of patients post TBI could give better quality of life.