Predictive Value of Phosphorylated Neurofilament H as A Marker of Brain Injury after Cardiac Arrest

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1 Med. J. Cairo Univ., Vol. 83, No. 2, June: , Predictive Value of Phosphorylated Neurofilament H as A Marker of Brain Injury after Cardiac Arrest MOHAMMED S. MOHAMMED, M.D.*; SAMEH K. EL-MARAGHI, M.D.*; NAEL SAMIR, M.D.** and HAMDY M. SABER, M.D.* The Department of Critical Care Medicine, Faculty of Medicine, Beni Suef* and Cairo** Universities, Egypt 205 Introduction ANOXIC brain insult is a sequalae of tissue hypoperfusion during cardiac arrest that may lead to varieties of neurological outcome in patients [1]. Specific proteins are expected to be released from neurons and their processes as a result of axonal damage following central nervous system injury post cardiac arrest. An assay that could reliably quantify the levels of these released proteins in blood might provide useful information about the degree of neuronal injury and could be potentially used to predict neurological outcome post cardiac arrest [2]. An ideal biomarker of axonal injury would have several properties; it should be expressed specifically in axons, it should be abundant enough so that it can be readily detectable after the significant dilution that occurs following release into blood, and it should be resistant to proteases so that it is not degraded prior to or following release [2]. Several biomarkers have been evaluated to assess brain damage after cardiac arrest with inconsistent results-for example, Neuronspecific Enolase (NSE), S-100B, and Glial Fibrillary Acidic Protein (GFAP) [3-5]. Several reasons suggest that the phosphorylated subunits of Neurofilaments, the major structural protein complexes of axons, are an ideal biomarker for brain injury following cardiac arrest. First, Neurofilament H is axon specific [6], and is known to be more resistant to calpain and other proteases than the other Neurofilament subunits [7]. In addition, Neurofilamets have been known for a long time to be highly immunogenic [8], and the multiple repeated phosphorylated sites are an excellent target for antibody-based assays [9].

2 206 Predictive Value of pnf-h as A Marker of Brain Injury Aim of the work: To assess the usefulness of the phophorylated form of Neurofilament H (pnf-h) as a marker of brain damage following in-hospital cardiac arrest and to correlate the plasma level of neurofilament H with outcome and prognosis of post cardiac arrest patients. Patients and Methods After approval of the study protocol by the local ethical committee 30 intra hospital cardiac arrest patients who were admitted to either the Critical Care Department of Beni Suef University or Cairo University in the period from August 2013 to September 2014 were randomly selected and an informed consent was signed from each patients close relative entailing all ethical and moral considerations, it is an observational prospective cohort study. Inclusion criteria: Thirty randomly selected patients who had intra hospital cardiac arrest and regained spontaneous circulation after successful CPR were included in the study. Exclusion criteria: The following patients were excluded from the study: Patients with renal impairment; creatinine clearance of less than 25ml/min before the cardiac arrest. History of dementia or any chronic neurological disease. Multi-organ failure syndrome. Neurologic trauma. Seizure activity. Methods: The study approved the Standard CPR protocol according the American heart association guidelines Following ROSC review of medical history, general exam and complete neurological examination, focused cardiac arrest data (suggested etiology -mode of arrest-time between arrest and starting the CPR-CPR details of relevance). Calculation of the patient's scores: A- Glasgow Coma Scale of patients was calculated to assess the neurological outcome on day 1 (GCS 1) and on day 3 post CPR (GCS2). B- Rankin scale was used on day 1 (Rankin 1) and on day 3 post CPR (Rankin 2) to assess patients' outcome. Each patient was given a number from 1 to 6 according to level of disability [10]. Table (1): Rankin Scale [10]. 6=Death. 5=Severe disability. 4=Moderately severe disability. 3=Moderate disability. 2=Slight disability. 1=No significant disability. - Bed ridden, incontinent, and requiring constant nursing care and attention. - Unable to walk without assistance, and unable to attend to own bodily needs without assistance. - Requiring some help, but able to walk without assistance. - Unable to carry out all previous activities but able to look after own affairs without assistance. - Able to carry out all usual duties and activities. C- All patients were followed by the Cerebral Performance Category scale (CPC) on day one (CPC 1) and after 3 days post CPR (CPC 2): CPC 1: Good cerebral performance. CPC 2: Moderate cerebral disability, independent. CPC 3: Severe cerebral disability, conscious but dependent. CPC 4: Coma. CPC 5: Death (11). Serum samples were drawn for pnf-h levels; samples were taken on day 1 post CPR (pnf-h1) then on day 3 (pnf-h2) post arrest and tested by the enzyme linked immunosorbent assay (ELISA) technique. Principle of assay: In the Bio Vendor Human Phosphorylated Neurofilament H ELISA, standards, quality controls and samples were incubated in microplate wells pre-coated with chicken polyclonal anti-pnf-h antibody. After 60 minutes of incubation and washing, detection rabbit polyclonal anti-pnf-h antibody was added and incubated with captured pnf- H for 60 minutes. After another washing, HRP conjugated antibody against rabbit antibody was added. After 60 minutes incubation and the last washing step, the remaining conjugate was allowed to react with the substrate solution. The reaction was stopped by addition of acidic solution and absorbance of the resulting yellow product was measured.

3 Mohammed S. Mohammed, et al. 207 Data analysis: Data were summarized by descriptive statistics using mean and standred deviation or frequency and percentage as appropriate. Mean values and standred deviation were compared using simple t- test (for two variables) or ANOVA test (for more than two variables). Percents are compared using chi-square (x 2 ) test. Pearson correlation was used for analysis of relation of bivaried. Linear repression was used to estimate the coefficient and linear equation involving one or more independent variables that best predict the value of dependent variable. A p-value <0.05 was considered statistically significant. ROC curve analysis was done to predict the cutoff points of the test variables that best predict the binary state of other variable. The software used in analysis is the statistical package SPSS (Self-Propelled Semi-Submersible) version 15. Results 1- Demographic, baseline clinical characteristics and cardiac arrest data: The mean age of all the studied population was (51.8±15.9 years); both males and females were represented in the ratio of 76.7% and 23.3% respectively. Risk factors of the studied population 53.3% were diabetic, 56.7% hypertensive, 46.6% dyslipidemic and 63.3% were smokers, all patients included in our study had in hospital cardiac arrest and all patients included were free of any neurological history or neurological deficit before arrest. 36.7% of patients were admitted to the ICU due to cardiac illness while 33.3% due to respiratory illness, 23.3% surgical related emergencies and gynecological related emergencies in 6.7%. During CPR (46%) of patients showed an initial rhythm of pulseless electrical activity or asystole while (20%) showed pulseless VT/VF. Only 33.3% of our patients received DC shock during CPR according to the ACLS guidelines. After ROSC 76.6% of our patients, inotropic support medications were needed, and 80% of patients were mechanically ventilated for more than 24 hours. 2- Scoring system data: The mean GCS on day one post CPR (GCS 1) was 7.43±3.6. While the mean GCS on day 3 (GCS 2) was 7.29±5.1 while the mean CPC on day one post CPR (CPC 1) was 3.46±0.77, while the mean CPC on day 3 (CPC 2) was 3.33±1.24. The mean Rankin scale on day one post CPR (Rankin 1) was 4.43±0.97; while the mean Rankin scale on day 3 (Rankin 2) was 3.96± Outcome analysis: Correlation between GCS and Neurofilament H levels: As shown by the scatter diagram Fig. (1) there were a strong negative correlation between the level of Neurofilament H and the Glasgow Coma Scale in patients with traumatic brain injury on day 1 (r= 0.56 with a p=0.001) also similar results on day 3 (r= , p<0.00 1) in other words, high pnf-h levels correlate with lower GCS [Fig. (1), (Table 2)]. Correlation between Cerebral Performance Category (CPC) scale and Neurofilament H levels: There were a statistically significant positive correlation between Neurofilament H and CPC scale on day 1 (r-value=0.522 and p-value=0.003) similarly on day 3 (r-value=0.845 and p-value <0.001) means high neurofilament levels were associated with a high CPC score and suggesting worse outcome [Fig. (2) and (Table 2)]. Correlation between pnf-h and rankin scale: There were a statistically significant positive correlation between Neurofilament H and Rankin scale on day 3 with ( r-value=0.814 and p-value >0.001) i.e., high Neurofilament levels were associated with a high Rankin scale (severe disability or death), while low levels of Neurofilament were associated with low Rankin scale (mild disability) [Fig. (3), (Table 2)]. However the correlation was non significant between Neurofilement H and Rankin scale on day 1 (r=0.353 and p-value=0.056). Receiver-Operating Characteristic (ROC) curves for Neurofilament H as a predictor of outcome: ROC curve was calculated for the use of pnf- H level on day 1 post cardiac arrest as a predictor for outcome. The Area Under Curve (AUC) for pnf-h to predict severe disability or death was 0.76 [(95%) confidence Interval ( )]. The optimal cutoff point of pnf-h to predict severe disability or death was 0.201ng/ml with sensitivity 85% and specificity of 70% with p-value of Fig. (4). Table (2): Correlation between pnf-h and GCS, CPC and Rankin. r-value p-value PNF-H and GCS Day 1 post arrest Day 3 post arrest <0.001 P NF-H and CPC Day 1 post arrest Day 3 post arrest <0.001 PNF-H and Rankin scale Day 1 post arrest Day 3 post arrest <0.001

4 208 Predictive Value of pnf-h as A Marker of Brain Injury GCS Rankin GCS- 2 GCS- (1 Linear GCS) (2 Linear GCS) (p-value <0.005) P-NFH ng/dl Fig. (1): Correlation between pnf-h1 level and Glasgow Coma Scale on day 1 (GCS1) and on day 3 (GCS3). 1 2 Rankin Linear 2 Rankin P-NFH2 ng/dl Fig. (3): Correlation between pnf-h and Rankin Scale on day 3. Discussion Post cardiac arrest is complex and dynamic multi-organ affection and is often referred to as the post-resuscitation syndrome [12]. The major cause of death in patients with Return of Spontaneous Circulation (ROSC) is ischemic brain damage, which evolves over several days [13]. In many patients who remain comatosed after cardiac arrest [14], a reliable assessment of neurological prognosis is therefore needed to be postponed for several days [15]. There has been a growing appreciation that many kinds of CNS injury and disease states are the result of axonal injury and degeneration. Accordingly, a convenient method of detecting ongoing axonal loss might be particularly useful experimentally and clinically [16]. Phosphorylated Neurofilament H (pnf-h) is a large protein with a molecular weight of kd depending on the degree of phosphorylation, it is axone specific, known to be more resistant to CPC Sensitivity ROC curve 1 CPC 2 CPC (1 Linear CPC) (2 Linear CPC) P-NFH ng/dl Fig. (2): Correlation between pnf-h level and Cerebral Performance category scale on day 1 (CPC 1) and on day 3 (CPC 3) Specificity Fig. (4): ROC curve analysis of pnf-h to detect disability on day 1. calpain and other proteases [6,7]. Also, pnf-h is highly immunogenic, and the multiple repeated phosphorylated sites are an excellent target for antibody-based assays [8,9]. These facts suggest that pnf-h might be a good candidate as a biomarker of axonal injury. Following neuro-axonal injury pnf-h is released into the extracellular fluid from where it can be measured using ELISA. From the extracellular fluid it diffuses into the CSF compartment and from the CSF, pnf-h reaches the blood via the blood CSF barrier at the lumbar level or it may diffuse directly through the cortical arachnoid villi to the blood stream [17]. In this study, we have demonstrated that pnf- H can be detected in the blood of patients with brain insult post-cardiac arrest. The presence of Neurofilaments in blood indicated damage of neurons after such injuries. The importance of pnf- H lies in the fact that it could be detected in the blood of post-cardiac arrest victims, which makes such marker a convenient and simple indicator of

5 Mohammed S. Mohammed, et al. 209 brain damage, in contrast to other markers measured in the CSF. Blood collection is more practical and safer than CSF sampling suggesting that analysis of blood for pnf-h could be a useful clinical tool to conveniently assess axonal damage [18]. The demographic data of our patients showed that mean age of the all studied population is (51.8 years ±15.9), males and females ratio were 76.7% and 23.3% respectively, both age and sex groups didn`t show any statistical significance when correlated to Neurofilament H levels or outcome with a p-value >0.05. This was in agreement with Malin Rundgren, et al., 2012 who studied Neurofilament H in 90 patients post cardiac arrest, included both males and females in his study in a ratio of 70% and 30% respectively with a median age of 64 years; it also showed no statistically significance correlation between age and sex groups and Neurofilament levels. We only included patients with in hospital cardiac arrest and excluded those with out-hospital cardiac arrest to be more accurate about the cause of cardiac arrest and be sure about the onset of arrest and time to initiation of CPR and adherence ACLS protocol to evaluate the impact on neurologic damage. However there were no statistically significant relations between Neurofilament H levels nor outcome when correlated with any of the risk factors with a p-value >0.05. Exploring the impact of Neurofilament H levels on neurological outcome, we correlated the pnf- H levels with Glasgow coma scale (GCS), we found a significant negative correlations between Neurofilament H and GCS, when measured on day one (r=0.56, p=0.001) and on day three post arrest (r= 0.92, p<0.001), in other words, higher levels of Neurofilament H were correlated with lower GCS and hence a worse neurological outcome this was in the same context with Malin Rundgren, et al who found significant increase in pnf-h levels in patients with poor outcome post cardiac arrest on day 1 and day 3 [19]. Also our results go hand in hand with study done by H Rosen, et al who studied CSF levels of Neurofilamint H in 22 patients with post cardiac arrest and found that Neurofilament H levels increased in cardiac arrest patients and were associated with poor outcome according to the Glasgow outcome scale (GCS=3,4,5) ( r=0.79, p<0.001) [20]. Our results also coincides with the study done by Moh Gonemi, et al who studied 90 patients with acute brain insults; and showed negative correlation between Neurofilament H levels and GCS on day 1 and day 7 in 30 patients with traumatic brain injury (r= 0.66, p<0.005) and (r= 0.78, p<0.005) respec- tively, and in 30 patients with cerebral hemorrhage (r= 0.56, p<0.005) and (r= 0.65, p<0.005) respectively, also it showed a significant inverse relationship between the GCS and the Neurofilamet levels in 30 patients with ischemic stroke on day 1 and on day 7 (r= 0.37, p<0.005) and (r= 0.5, p<0.005), respectively [21]. Aiming to assess outcome and disability, Cerebral Performance Category (CPC) scale were used and we found that patients with higher levels of Neurofilament H on day 1 post arrest and on day 3 showed a greater CPC scale score hence liable for greater disability and poor outcome (CPC= 3,4,5), (r=0.52, p=0.003) and (r=0.84, p<0.001) respectively this was in agreement with Rundgren, et al., who also studied the cerebral performance category to assess outcome. They found that plasma Neurofilamint H levels were significantly higher 2 hours and 36 hours after cardiac arrest in patients with poor outcome (CPC =3,4,5) (0.28ng/mL, p- value=0.002) and (0.5 ng/ml, p-value <0.001) respectively, compared to those with good outcome (0ng/ml, p-value=0.016) and (0.1ng/ml, p<0.005) respectively [19]. In our study Rankin scale was also calculated to assess general outcome, we found that patients with higher levels of Neurofilament H on day 3 post arrest showed a greater Rankin scale (Rankin =5,6) hence a greater disability and poor outcome (r=0.81, p<0.001). Rosen, et al., used other scores than CPC and RANKIN used in our study to follow-up performance and disability he found that patients with low performance at a mini mental state examination (MMSE) at 1 year follow-up had the highest Neurofilement levels [20]. Our findings were similar to Gonemi, et al., who studied 90 patients with acute brain insults and used Rankin scale to assess disability and outcome, they found that patients with higher levels of Neurofilament H on day 1 and on day 7 post admission showed a greater Rankin scale (Rankin 5,6) and hence a greater disability and poor outcome (p<0.005) [21]. Our results regarding the increase of Neurofilamint H in brain injury agreed with that of Singh and Yan, which showed that Serum Neurofilamets levels were significantly elevated in stroke patients compared to healthy controls. Significant associations were found between the Neurofilamets H and stroke severity, size and outcome. Blood Neurofilament H levels correlated with outcome and rise during the weeks after stroke [22].

6 210 Predictive Value of pnf-h as A Marker of Brain Injury Regarding the increase of Neurofilamint H level as a results of axonal injury, similar to our results Hayakawa and Okazaki, stated that Plasma Neurofilamet H levels were elevated in accordance with the severity of Spinal cord injury and reflected a greater magnitude of axonal damage. They concluded that Neurofilamet H is a potential biomarker to independently distinguish patients with complete Spinal cord injury from patients with incomplete Spinal cord injury [23]. ROC curve analysis for p NF-H levels on day 1 post cardiac arrest as a predictor for outcome. The Area Under Curve (AUC) for pnf-h to predict severe disability or death was 0.76 [95% confidence Interval ( )]. The optimal cutoff point of pnf-h to predict severe disability or death was 0.201ng/ml with sensitivity 85% and specificity of 70% with p-value of Similary; Gonemi et al., was able to identify a cut off point for pnf- H level on admission to predict severe disability or death which was 35pg/ml with sensitivity 82.1% and specificity 78.4% [21]. However Rundgren, et al., were not able to identify any cut-off levels or predictive values to be recommended for individual patients due to a considerable data overlap [19]. Several other proteins have been proposed as potential biomarkers in post cardiac arrest for example, Martens, et al., reported that serum S 100B concentrations at 24h after arrest were significantly higher in patients remaining comatosed after cardiac arrest than those regaining consciousness [24]. Several clinical studies have also shown that increased serum concentration of S 100B predicted extensive post anoxic brain damage; however, different cutoffs have been proposed, varying from 0.2 to 1.5mg/l [24,26], depending also on the different time of sampling and on the dosage methods. However, S 100B within the first 2 days after cardiac arrest showed a median False Positive Rate of 2-5% in identifying patients with poor outcome [25,26], and was initially considered as a poor prognostic indicator [27]. As S 100B elevation after cardiac arrest could be due to extracerebral release from adipocytes and chondrocytes, perhaps as a result of chest compressions [28]. Previous studies have also examined serum levels of Neuron Specific Enolase (NSE) as a marker of post cardiac arrest state in humans, Roine, et al first reported high NSE values in nonsurvivors after cardiac arrest [28]. Martens, et al., also showed higher serum NSE levels at 24h after arrest in patients with persistent post anoxic coma compared to those with subsequent recovery of normal brain function [24]. Hemolysis of blood samples may lead to a potential misclassification of patients' prognosis according to NSE levels [30] ; this event which may occur when using intracorporeal or extracorporeal assisting devices for left ventricular dysfunction, such as the intra-aortic balloon pump conterpulsation or the extracorporeal membrane oxygenation, which may cause mechanical destruction of blood cells [30] ; the use of such devices is largely increased in cardiogenic shock following cardiac arrest, NSE levels may be significantly influenced and of limited prognostic value in these conditions [30]. Hence, it seems that Neurofilament H is a useful marker not only in cardiac arrest patients, but also in different forms of brain injury as in stroke and traumatic brain injury. Conclusion: Levels of Neurofilament H correspond to the severity of injury as shown by the presence of significant correlations between Neurofilament levels and GCS, CPC and Rankin scale. Neurofilament H levels can be used as a prognostic marker to detect the degree of disability and death in patients with ROSC after cardiac arrest. Neurofilament H seems to be a promising marker for diagnosing patients with brain insults and for the short term follow-up of such patients. Further studies will be needed to complement our results in larger patients samples and more importantly to elucidate other non-cranial causes of increased Neurofilament H level in serum. References 1- BELAYEV L., BUSTO R., ZHAO W., et al.: Quantitative evaluation of blood-brain barrier permeability following middle cerebral artery occlusion in rats. Brain Res., Nov. 11, 739 (1-2): 88-96, BUKI A. and POVLISHOCK J.T.: All roads lead to disconnection-traumatic axonal injury revisited. Acta. 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8 212 Predictive Value of pnf-h as A Marker of Brain Injury