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Relationship between plasma leptin levels and clinical outcomes of pediatric traumatic brain injury
Peptides 35 (2012) 166–171
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Peptides journal homepage: www.elsevier.com/locate/peptides
Relationship between plasma leptin levels and clinical outcomes of pediatric traumatic brain injury夽 Chao Lin, Shou-Jiang Huang ∗ , Ning Wang, Zhi-Peng Shen Department of Neurosurgery, The Children’s Hospital, School of Medicine, Zhejiang University, 57 Zhugan Lane, Hangzhou 310000, China
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Article history: Received 25 February 2012 Received in revised form 25 March 2012 Accepted 26 March 2012 Available online 3 April 2012 Keywords: Leptin Traumatic brain injury Functional outcome Mortality
a b s t r a c t High plasma leptin level has been associated with mortality after adult intracerebral hemorrhage. The present study was undertaken to investigate the plasma leptin concentrations in children with traumatic brain injury and to analyze the correlation of leptin with pediatric traumatic brain injury outcome. Plasma leptin concentration of eighty-nine healthy children and 142 children with acute severe traumatic brain injury was measured by enzyme-linked immunosorbent assay. Twenty-six patients (18.3%) died and 42 patients (29.6%) had an unfavorable outcome (Glasgow outcome scale score of 1–3) at 6 months after traumatic brain injury. Upon admission, plasma leptin level in patients was substantially higher than that in healthy controls. A forward stepwise logistic regression selected plasma leptin level as an independent predictor for 6-month mortality and unfavorable outcome of patients. A receiver operating characteristic curve analysis showed plasma leptin level better predicted 6-month mortality and unfavorable outcome. The prognostic value of leptin was similar to that of Glasgow Coma scale score for 6-month clinical outcomes. Thus, plasma leptin level represents a novel biomarker for predicting 6-month clinical outcome in children with traumatic brain injury. © 2012 Elsevier Inc. All rights reserved.
1. Introduction The endocrine hormone leptin is principally, but not exclusively, derived from white adipose tissue [2,15]. The circulating levels of this hormone vary during the day, but are mainly determined by body fat content and also feeding status [19]. Leptin enters the brain via saturable transport across the blood brain barrier [6]. Additionally, leptin mRNA is selectively transcribed in specific areas of the brain and pituitary in rat, pig, sheep and human [23,34]. It is interesting that abnormalities in brain development are present in ob/ob mice, suggesting that leptin is required for normal neuronal and glial maturation [1]. In addition, brain cortex leptin mRNA expression and serum leptin level are up-regulated in ischemic mouse brain, as well as in rat brain with traumatic brain injury [8,32,33]. Previous clinical study has shown that elevated leptin plasma levels predict cerebral hemorrhagic stroke independently of traditional risk factors [28,29]. Recent studies in adults have demonstrated that leptin is increased after intracerebral hemorrhage [11]; in these groups of patients, high leptin levels were highly predictive for poor function outcome and mortality.
夽 Institution at which the work was performed: The Children’s Hospital, School of Medicine, Zhejiang University. ∗ Corresponding author. Tel.: +86 0571 87061007. E-mail address: [email protected] (S.-J. Huang). 0196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2012.03.024
However, at present there is a paucity of data available on circulating plasma leptin concentrations in pediatric traumatic brain injury (TBI) patients. The present study was undertaken to investigate the plasma leptin concentrations in children with TBI and to analyze the correlation of leptin with pediatric TBI outcome. 2. Materials and methods 2.1. Study population This study was conducted in the Department of Neurosurgery, The Children’s Hospital, School of Medicine, Zhejiang University. During the period from January 2009 to January 2011, all isolated head trauma children (aged below 15 years) with a postresuscitation Glasgow Coma scale (GCS) score of 8 or less were initially assessed. Exclusion criteria were the disagreement of the parents (legal representatives) with participation of their children in this study, recent infection, admission time > 6 h, previous head trauma, and history of seizure. Children were eligible as controls if they presented to our hospital and had blood collected as part of well-child care (e.g., as part of a 1 year well-child examination which includes measurement of hemoglobin) between January 2010 and May 2010. Exclusion criteria were the disagreement of the parents (legal representatives) with participation of their children in this study, recent infection, previous head trauma, and history of seizure.
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The study protocol and informed consent approach were approved by the Ethics Committee of the Children’s Hospital, School of Medicine, Zhejiang University before implementation. The parents provided written informed consent for their children to participate in this trial. 2.2. Clinical and radiological assessment At admission, we recorded age, gender, vital signs (heart rate, respiratory rate, systolic and diastolic blood pressure), body temperature in ◦ C, GCS score [30], pediatric trauma score [31], injury severity score [5]. All computerized tomography (CT) scans were performed according to the neuroradiology department protocol. Investigators who read them were blinded to clinical information. Midline shift >5 mm, abnormal basal cisterns (compressed or absent cisterns) and traumatic subarachnoid hemorrhage were recorded. CT classification was performed using Traumatic Coma Data Bank criteria on initial postresuscitation CT scan according to the method of Marshall et al. [20]. 2.3. Patient management After diagnostic assessment and/or surgery, all patients were transferred to the pediatric intensive care unit and received intensive care, including mechanical ventilation, and hemodynamic monitoring. Intracranial hypertension was treated progressively by a standard step-wise protocol that included sedation, paralysis, mild hyperventilation during prolonged, refractory intracranial hypertension, osmotherapy with mannitol, and use of barbiturates. 2.4. Immunoassay method The informed consents were obtained from their parents in all cases before the blood were collected. Venous blood was drawn at study entry in the control group and on admission in the patients. The blood samples were immediately placed into sterile EDTA test tubes and centrifuged at 3000 × g for 30 min at 4 ◦ C to collect plasma. Plasma was stored at −70 ◦ C until assayed. The concentration of leptin in plasma was analyzed by enzymelinked immunosorbent assay using commercial kits (R&D Systems, Minneapolis, MN, USA) in accordance with the manufactures’ instructions. The person carrying out the assays was completely blinded to the clinical information.
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Table 1 The main baseline demographic, clinical, and radiologic characteristics for 142 head trauma children. Characteristics Sex (male/female) Age (month) GCS score on admission Pediatric trauma score on admission Injury severity score on admission Pupils unreactive on admission CT classification 5 or 6 Abnormal cisterns on initial CT scan Midline shift >5 mm on initial CT scan Traumatic SAH on initial CT scan Mechanical ventilation Intracranial surgery in 1st 24 h Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Heart rate (beats/min) Body temperature (◦ C) Respiratory rate (respirations/min) Blood glucose level (mmol/L) Plasma C-reactive protein level (mg/L) Plasma leptin level (ng/mL)
96/46 72 (64) 6 (3) 5 (3) 27 (3) 56 (39.4%) 68 (47.9%) 59 (41.6%) 64 (45.1%) 75 (52.8%) 121 (85.2%) 72 (50.7%) 3.1 ± 1.4 3.5 ± 1.6 95.9 ± 20.0 60.6 ± 11.9 107.8 ± 21.3 37.1 ± 0.7 24.7 ± 4.3 9.6 ± 4.4 6.8 ± 2.6 12.5 ± 6.9
The categorical variables are presented as counts (percentage), and the continuous variables are presented as mean ± standard deviation if normally distributed or median (interquartile range) if not normally distributed. GCS, Glasgow Coma scale; CT, computerized tomography; SAH, subarachnoid hemorrhage.
Fisher exact test for categorical variables, and by Student’s t or Mann–Whitney U test for continuous variables. Receiver operating characteristic (ROC) curves were configured to establish cutoff points of plasma leptin level that optimally predicted mortality and functional outcome. Multivariable logistic regression analyses were performed to determine factors that could be considered as independent predictors of mortality and functional outcome, adjusted by confounding variables according to the results of the univariate analysis. Variables showing P < 0.1 in univariate analysis were included in the multivariate model. The logistic regression results are presented as odds ratio (OR) and 95% confidence interval (CI). A P value of <0.05 was considered significant for all tests. 3. Results 3.1. Patient characteristics
Participants were followed up until death or completion of 6 months after head trauma. The end points were unfavorable outcome and death after 6 months. The functional outcome was defined by Glasgow outcome scale (GOS) score. GOS was defined as follows: 1 = death; 2 = persistent vegetative state; 3 = severe disability; 4 = moderate disability; 5 = good recovery [16]. GOS scores were dichotomized in favorable and unfavorable outcomes (GOS of 4–5 vs. GOS of 1–3). The person who determined the outcome was completely blinded to the clinical information.
During the study period, a total of 168 consecutive head trauma children were initially evaluated. Of these, 26 patients were excluded for the following reasons shown in Fig. 1, and 142 patients were finally included in the analysis. The main baseline demographic, clinical, and radiologic characteristics of the series are summarized in Table 1. Overall, 26 patients (18.3%) died and 42 patients (29.6%) had an unfavorable outcome at 6 months. 89 healthy children were eligible as controls. The intergroup differences in the age and sex were not statistically significant. Plasma leptin level was obviously higher in patients than in healthy children (12.5 ± 6.9 ng/mL vs. 3.1 ± 0.9 ng/mL, P < 0.001).
2.6. Statistical analysis
3.2. Impact of plasma leptin level on 6-month mortality
Statistical analysis was done using the SPSS 12.0 statistical package (SPSS Inc., Chicago, IL, USA) and MedCalc 9.6.4.0 (MedCalc Software, Mariakerke, Belgium). The categorical variables are presented as percentages, and the continuous variables are presented as mean ± standard deviation if normally distributed or median (interquartile range) if not normally distributed. Statistical significance for intergroup differences was assessed by chi-square or
Potential predictors of 6-month mortality are shown in Table 2. Patients who experienced 6-month mortality had higher glucose, C-reactive protein and leptin level, lower GCS score, and more frequently showed unreactive pupils, CT classification 5 or 6, abnormal cisterns, midline shift >5 mm and traumatic subarachnoid hemorrhage on initial CT scan, and need more mechanical ventilation. Multivariate logistic regression analysis showed that variables
2.5. End point
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Fig. 1. Graph documenting patients’ entry into the study from screening.
Table 2 The main baseline demographic, clinical, and radiologic characteristics associated with 6-month mortality for 142 head trauma children.
Number Sex (male/female) Age (month) GCS score on admission Pediatric trauma score on admission Injury severity score on admission Pupils unreactive on admission CT classification 5 or 6 Abnormal cisterns on initial CT scan Midline shift >5 mm on initial CT scan Traumatic SAH on initial CT scan Mechanical ventilation Intracranial surgery in 1st 24 h Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Heart rate (beats/min) Body temperature (◦ C) Respiratory rate (respirations/min) Blood glucose level (mmol/L) Plasma C-reactive protein level (mg/L) Plasma leptin level (ng/mL)
Nonsurvival Group
Survival Group
P value
26 (18.3%) 19/7 72 (60) 4 (2) 6 (3) 27 (6) 23 (88.5%) 22 (84.6%) 21 (80.8%) 19 (73.1%) 20 (76.9%) 26 (100.0%) 16 (61.5%) 3.2 ± 1.5 3.7 ± 1.7 98.5 ± 18.4 62.7 ± 13.6 107.0 ± 15.9 37.0 ± 0.7 23.6 ± 3.5 11.6 ± 4.0 7.8 ± 2.7 20.1 ± 5.1
116 (81.7%) 77/39 60 (65) 6 (3) 5 (4) 27 (3) 33 (28.5%) 46 (39.7%) 38 (32.8%) 45 (38.8%) 55 (47.4%) 95 (81.9%) 56 (48.3%) 3.0 ± 1.3 3.4 ± 1.4 95.3 ± 20.3 60.2 ± 11.5 108.0 ± 22.3 37.1 ± 0.8 25.0 ± 4.4 9.2 ± 4.4 6.5 ± 2.6 10.9 ± 6.3
0.510 0.310 <0.001 0.380 0.856 <0.001 <0.001 <0.001 0.001 0.006 0.014 0.221 0.165 0.242 0.459 0.334 0.823 0.696 0.130 0.012 0.026 <0.001
The categorical variables are presented as counts (percentage), and the continuous variables are presented as mean ± standard deviation if normally distributed or median (interquartile range) if not normally distributed. Statistical significance for intergroup differences was assessed by chi-square or Fisher exact test for categorical variables, and by Student’s t or Mann–Whitney U test for continuous variables. GCS, Glasgow Coma scale; CT, computerized tomography; SAH, subarachnoid hemorrhage.
Table 3 Receiver operating characteristic curve analysis of factors predicting 6-month mortality for 142 head trauma children.
Criterion Area under curve (95% CI) Sensitivity (95% CI) Specificity (95% CI) Positive likelihood ratio (95% CI) Negative likelihood ratio (95% CI) Positive predictive value (95% CI) Negative predictive value (95% CI) P value GCS indicates Glasgow Coma scale; CI, confidence interval.
Leptin
GCS score
>14.4 ng/mL 0.859 (0.791–0.912) 96.2 (80.3–99.4) 65.6 (56.1–74.1) 2.79 (2.4–3.2) 0.059 (0.008–0.4) 38.5 (26.7–51.4) 98.7 (92.9–99.8) Reference
<5 0.905 (0.845–0.948) 92.3 (74.8–98.8) 70.7 (61.5–78.8) 3.15 (2.7–3.7) 0.11 (0.03–0.4) 41.4 (28.6–55.1) 97.6 (91.6–99.6) 0.303
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4. Discussion
Fig. 2. Graph showing receiver operating characteristic curve analysis of plasma leptin level for 6-month mortality.
independently related to 6-month mortality were plasma leptin level (OR 1.225, 95% CI 1.102–1.519, P = 0.006) and GCS score (OR 0.374, 95% CI 0.232–0.671, P = 0.001). ROC curves identified cutoff points for plasma leptin level on admission as the value that better predicted 6-month mortality (Fig. 2). The predictive value of the leptin concentration was thus similar to that of GCS score (Table 3). 3.3. Impact of plasma leptin level on the unfavorable outcome at 6 months Potential predictors of 6-month unfavorable outcome are shown in Table 4. Patients who experienced 6-month unfavorable outcome had higher glucose, C-reactive protein and leptin level, lower GCS score, and more frequently showed unreactive pupils, CT classification 5 or 6, abnormal cisterns, midline shift >5 mm and traumatic subarachnoid hemorrhage on initial CT scan, and need more mechanical ventilation. Multivariate logistic regression analysis showed that variables independently related to 6-month unfavorable outcome were plasma leptin level (OR 1.340, 95% CI 1.162–1.678, P = 0.002) and GCS score (OR 0.331, 95% CI 0.204–0.629, P = 0.001). ROC curves identified cutoff points for plasma leptin level on admission as the value that better predicted 6-month unfavorable outcome (Fig. 3). The predictive value of the leptin concentration was thus similar to that of GCS score (Table 5).
Fig. 3. Graph showing receiver operating characteristic curve analysis of plasma leptin level for 6-month functional outcome.
This study was conducted to determine if leptin is increased in the circulation of children with TBI and whether this increment correlates with 6-month clinical outcomes in these pediatric patients. The admission plasma leptin levels were indeed significantly increased in all patients compared with healthy subjects. Furthermore, an admission plasma leptin level was identified as a reliable and independent marker to predict patients at risk of 6month poor clinical outcomes. Importantly, the prognostic value of leptin was similar to that of GCS score for 6-month clinical outcomes, substantiating its potential as a new prognostic biomarker. Originally thought to be a satiety factor, leptin is a pleiotropic molecule [25]. Since its cloning in 1994, leptin’s role in regulating inflammatory response has become increasing evident. Both the structure of leptin and that of its receptor suggest that leptin might be classified as a cytokine [14]. The secondary structure of leptin has similarities to the long-chain helical cytokines family, which includes interleukin-6, interleukin-1, leukemia inhibitory factor, and ciliary neurotrophic factor [36]. Leptin receptors are encoded by the diabetes gene and homologous to the gp-130 signaltransducing subunit of the interleukin-6-type cytokine receptors [7]. Indeed, leptin regulates several cytokine secretion patterns. It has been shown that different inflammatory stimuli, including interleukin-1, interleukin-6, tumor necrosis factor-alpha or lipopolysaccharide, regulate leptin mRNA expression as well as circulating leptin levels [13,24,27]. Furthermore, leptin is produced by inflammatory-regulatory cells [26]. Conversely, leptin enhances the production of C-reactive protein, tumor necrosis-alpha and interkeulin-6, suggesting that leptin expression could participate in the inflammatory process [10,17,35]. Actually, the increase of leptin production that occurs during infection and inflammation strongly suggests that leptin is a part of the cytokines loop [4,12]. In central nervous system, brain cortex leptin mRNA expression and serum leptin level are up-regulated in ischemic mouse brain, as well as in rat brain with traumatic brain injury [8,32,33]. Concerning experimental autoimmune encephalomyelitis, it has been shown that leptin-deficient ob/ob mice are resistance to the development of this model of multiple sclerosis. This resistance is abolished by the administration of leptin, which is accompanied by a switch from a TH2 to TH1 pattern of cytokine release [22]. In addition, it has been noticed that the onset of multiple sclerosis is preceded by an increase of circulating leptin [21]. Noteworthy, it has been shown that leptin is expressed by both macrophages and T-cells infiltrated into the central nervous system during autoimmune encephalomyelitis [26]. This interesting report indicates that leptin is produced by immune cells during acute autoimmune encephalomyelitis. Recently, it is evidenced that administration of leptin increased levels of interleukin-1 beta in the hypothalamus in the rat [18]. It is suggested that leptin could participate in the development of central nervous system-inflammatory diseases. Brain injury is known to increase expression of several inflammatory cytokines, such as interleukin-1, interleukin-6, and tumor necrosis factor-alpha [3,9], which are capable of inducing leptin gene expression [13,24,27]. In intracerebral hemorrhage, plasma leptin level was found to be related to C-reactive protein level [11]. These findings suggested leptin may contribute to the inflammatory process of brain injury. This study found increased plasma leptin level in children after TBI in association with a worse clinical outcome. To our knowledge, this is the first time that the relationship of plasma leptin level with outcome has been investigated soon after TBI. In this study, plasma leptin level was highly associated with 6-month clinical outcome in children with TBI. Therefore, the determination of leptin in the plasma of pediatric patients on admission provides the ability to distinguish between patients with 6-month good and bad outcome.
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Table 4 The main baseline demographic, clinical, and radiologic characteristics associated with 6-month unfavorable outcome for 142 head trauma children.
Number Sex (male/female) Age (month) GCS score on admission Pediatric trauma score on admission Injury severity score on admission Pupils unreactive on admission CT classification 5 or 6 Abnormal cisterns on initial CT scan Midline shift > 5 mm on initial CT scan Traumatic SAH on initial CT scan Mechanical ventilation Intracranial surgery in 1st 24 h Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Heart rate (beats/min) Body temperature (◦ C) Respiratory rate (respirations/min) Blood glucose level (mmol/L) Plasma C-reactive protein level (mg/L) Plasma leptin level (ng/mL)
Unfavorable outcome
Favorable outcome
P value
42 (29.6%) 29/13 72 (85) 4 (1) 5 (3) 27 (6) 30 (71.4%) 31 (73.8%) 29 (69.0%) 27 (64.3%) 30 (71.4%) 42 (100.0%) 25 (59.5%) 3.4 ± 1.6 3.8 ± 1.7 98.3 ± 20.7 61.7 ± 13.2 110.9 ± 17.1 37.0 ± 0.7 24.0 ± 4.4 10.8 ± 4.0 7.9 ± 2.9 19.6 ± 4.4
100 (70.4%) 67/33 72 (60) 7 (2) 6 (4) 27 (3) 26 (26.0%) 37 (37.0%) 30 (30.0%) 37 (37.0%) 45 (45.0%) 79 (79.0%) 47 (47.0%) 3.0 ± 1.3 3.4 ± 1.5 94.9 ± 19.7 60.2 ± 11.3 106.5 ± 2.7 37.1 ± 0.8 25 ± 4.2 9.1 ± 4.5 6.3 ± 2.4 9.6 ± 4.7
0.812 0.588 <0.001 0.292 0.552 <0.001 <0.001 <0.001 0.003 0.004 0.001 0.173 0.103 0.182 0.351 0.495 0.266 0.613 0.191 0.037 0.002 <0.001
The categorical variables are presented as counts (percentage), and the continuous variables are presented as mean ± standard deviation if normally distributed or median (interquartile range) if not normally distributed. Statistical significance for intergroup differences was assessed by chi-square or Fisher exact test for categorical variables, and by Student’s t or Mann–Whitney U test for continuous variables. GCS, Glasgow Coma scale; CT, computerized tomography; SAH, subarachnoid hemorrhage.
Table 5 Receiver operating characteristic curve analysis of factors predicting 6-month unfavorable outcome for 142 head trauma children.
Criterion Area under curve (95% CI) Sensitivity (95% CI) Specificity (95% CI) Positive likelihood ratio (95% CI) Negative likelihood ratio (95% CI) Positive predictive value (95% CI) Negative predictive value (95% CI) P value
Leptin
GCS score
>14.1 ng/mL 0.891 (0.827–0.937) 92.9 (80.5–98.4) 72.0 (62.1–80.5) 3.32 (2.9–3.8) 0.099 (0.03–0.3) 58.2 (45.5–70.1) 96.0 (88.7–99.1) Reference
<5 0.936 (0.883–0.970) 90.5 (77.4–97.3) 80.0 (70.8–87.3) 4.52 (3.9–5.2) 0.12 (0.04–0.3) 65.5 (51.9–77.5) 95.2 (88.2–98.7) 0.192
GCS, Glasgow Coma scale; CI, confidence interval.
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Relationship between plasma leptin levels and clinical outcomes of pediatric traumatic brain injury夽 Chao Lin, Shou-Jiang Huang ∗ , Ning Wang, Zhi-Peng Shen Department of Neurosurgery, The Children’s Hospital, School of Medicine, Zhejiang University, 57 Zhugan Lane, Hangzhou 310000, China
a r t i c l e
i n f o
Article history: Received 25 February 2012 Received in revised form 25 March 2012 Accepted 26 March 2012 Available online 3 April 2012 Keywords: Leptin Traumatic brain injury Functional outcome Mortality
a b s t r a c t High plasma leptin level has been associated with mortality after adult intracerebral hemorrhage. The present study was undertaken to investigate the plasma leptin concentrations in children with traumatic brain injury and to analyze the correlation of leptin with pediatric traumatic brain injury outcome. Plasma leptin concentration of eighty-nine healthy children and 142 children with acute severe traumatic brain injury was measured by enzyme-linked immunosorbent assay. Twenty-six patients (18.3%) died and 42 patients (29.6%) had an unfavorable outcome (Glasgow outcome scale score of 1–3) at 6 months after traumatic brain injury. Upon admission, plasma leptin level in patients was substantially higher than that in healthy controls. A forward stepwise logistic regression selected plasma leptin level as an independent predictor for 6-month mortality and unfavorable outcome of patients. A receiver operating characteristic curve analysis showed plasma leptin level better predicted 6-month mortality and unfavorable outcome. The prognostic value of leptin was similar to that of Glasgow Coma scale score for 6-month clinical outcomes. Thus, plasma leptin level represents a novel biomarker for predicting 6-month clinical outcome in children with traumatic brain injury. © 2012 Elsevier Inc. All rights reserved.
1. Introduction The endocrine hormone leptin is principally, but not exclusively, derived from white adipose tissue [2,15]. The circulating levels of this hormone vary during the day, but are mainly determined by body fat content and also feeding status [19]. Leptin enters the brain via saturable transport across the blood brain barrier [6]. Additionally, leptin mRNA is selectively transcribed in specific areas of the brain and pituitary in rat, pig, sheep and human [23,34]. It is interesting that abnormalities in brain development are present in ob/ob mice, suggesting that leptin is required for normal neuronal and glial maturation [1]. In addition, brain cortex leptin mRNA expression and serum leptin level are up-regulated in ischemic mouse brain, as well as in rat brain with traumatic brain injury [8,32,33]. Previous clinical study has shown that elevated leptin plasma levels predict cerebral hemorrhagic stroke independently of traditional risk factors [28,29]. Recent studies in adults have demonstrated that leptin is increased after intracerebral hemorrhage [11]; in these groups of patients, high leptin levels were highly predictive for poor function outcome and mortality.
夽 Institution at which the work was performed: The Children’s Hospital, School of Medicine, Zhejiang University. ∗ Corresponding author. Tel.: +86 0571 87061007. E-mail address: [email protected] (S.-J. Huang). 0196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2012.03.024
However, at present there is a paucity of data available on circulating plasma leptin concentrations in pediatric traumatic brain injury (TBI) patients. The present study was undertaken to investigate the plasma leptin concentrations in children with TBI and to analyze the correlation of leptin with pediatric TBI outcome. 2. Materials and methods 2.1. Study population This study was conducted in the Department of Neurosurgery, The Children’s Hospital, School of Medicine, Zhejiang University. During the period from January 2009 to January 2011, all isolated head trauma children (aged below 15 years) with a postresuscitation Glasgow Coma scale (GCS) score of 8 or less were initially assessed. Exclusion criteria were the disagreement of the parents (legal representatives) with participation of their children in this study, recent infection, admission time > 6 h, previous head trauma, and history of seizure. Children were eligible as controls if they presented to our hospital and had blood collected as part of well-child care (e.g., as part of a 1 year well-child examination which includes measurement of hemoglobin) between January 2010 and May 2010. Exclusion criteria were the disagreement of the parents (legal representatives) with participation of their children in this study, recent infection, previous head trauma, and history of seizure.
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The study protocol and informed consent approach were approved by the Ethics Committee of the Children’s Hospital, School of Medicine, Zhejiang University before implementation. The parents provided written informed consent for their children to participate in this trial. 2.2. Clinical and radiological assessment At admission, we recorded age, gender, vital signs (heart rate, respiratory rate, systolic and diastolic blood pressure), body temperature in ◦ C, GCS score [30], pediatric trauma score [31], injury severity score [5]. All computerized tomography (CT) scans were performed according to the neuroradiology department protocol. Investigators who read them were blinded to clinical information. Midline shift >5 mm, abnormal basal cisterns (compressed or absent cisterns) and traumatic subarachnoid hemorrhage were recorded. CT classification was performed using Traumatic Coma Data Bank criteria on initial postresuscitation CT scan according to the method of Marshall et al. [20]. 2.3. Patient management After diagnostic assessment and/or surgery, all patients were transferred to the pediatric intensive care unit and received intensive care, including mechanical ventilation, and hemodynamic monitoring. Intracranial hypertension was treated progressively by a standard step-wise protocol that included sedation, paralysis, mild hyperventilation during prolonged, refractory intracranial hypertension, osmotherapy with mannitol, and use of barbiturates. 2.4. Immunoassay method The informed consents were obtained from their parents in all cases before the blood were collected. Venous blood was drawn at study entry in the control group and on admission in the patients. The blood samples were immediately placed into sterile EDTA test tubes and centrifuged at 3000 × g for 30 min at 4 ◦ C to collect plasma. Plasma was stored at −70 ◦ C until assayed. The concentration of leptin in plasma was analyzed by enzymelinked immunosorbent assay using commercial kits (R&D Systems, Minneapolis, MN, USA) in accordance with the manufactures’ instructions. The person carrying out the assays was completely blinded to the clinical information.
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Table 1 The main baseline demographic, clinical, and radiologic characteristics for 142 head trauma children. Characteristics Sex (male/female) Age (month) GCS score on admission Pediatric trauma score on admission Injury severity score on admission Pupils unreactive on admission CT classification 5 or 6 Abnormal cisterns on initial CT scan Midline shift >5 mm on initial CT scan Traumatic SAH on initial CT scan Mechanical ventilation Intracranial surgery in 1st 24 h Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Heart rate (beats/min) Body temperature (◦ C) Respiratory rate (respirations/min) Blood glucose level (mmol/L) Plasma C-reactive protein level (mg/L) Plasma leptin level (ng/mL)
96/46 72 (64) 6 (3) 5 (3) 27 (3) 56 (39.4%) 68 (47.9%) 59 (41.6%) 64 (45.1%) 75 (52.8%) 121 (85.2%) 72 (50.7%) 3.1 ± 1.4 3.5 ± 1.6 95.9 ± 20.0 60.6 ± 11.9 107.8 ± 21.3 37.1 ± 0.7 24.7 ± 4.3 9.6 ± 4.4 6.8 ± 2.6 12.5 ± 6.9
The categorical variables are presented as counts (percentage), and the continuous variables are presented as mean ± standard deviation if normally distributed or median (interquartile range) if not normally distributed. GCS, Glasgow Coma scale; CT, computerized tomography; SAH, subarachnoid hemorrhage.
Fisher exact test for categorical variables, and by Student’s t or Mann–Whitney U test for continuous variables. Receiver operating characteristic (ROC) curves were configured to establish cutoff points of plasma leptin level that optimally predicted mortality and functional outcome. Multivariable logistic regression analyses were performed to determine factors that could be considered as independent predictors of mortality and functional outcome, adjusted by confounding variables according to the results of the univariate analysis. Variables showing P < 0.1 in univariate analysis were included in the multivariate model. The logistic regression results are presented as odds ratio (OR) and 95% confidence interval (CI). A P value of <0.05 was considered significant for all tests. 3. Results 3.1. Patient characteristics
Participants were followed up until death or completion of 6 months after head trauma. The end points were unfavorable outcome and death after 6 months. The functional outcome was defined by Glasgow outcome scale (GOS) score. GOS was defined as follows: 1 = death; 2 = persistent vegetative state; 3 = severe disability; 4 = moderate disability; 5 = good recovery [16]. GOS scores were dichotomized in favorable and unfavorable outcomes (GOS of 4–5 vs. GOS of 1–3). The person who determined the outcome was completely blinded to the clinical information.
During the study period, a total of 168 consecutive head trauma children were initially evaluated. Of these, 26 patients were excluded for the following reasons shown in Fig. 1, and 142 patients were finally included in the analysis. The main baseline demographic, clinical, and radiologic characteristics of the series are summarized in Table 1. Overall, 26 patients (18.3%) died and 42 patients (29.6%) had an unfavorable outcome at 6 months. 89 healthy children were eligible as controls. The intergroup differences in the age and sex were not statistically significant. Plasma leptin level was obviously higher in patients than in healthy children (12.5 ± 6.9 ng/mL vs. 3.1 ± 0.9 ng/mL, P < 0.001).
2.6. Statistical analysis
3.2. Impact of plasma leptin level on 6-month mortality
Statistical analysis was done using the SPSS 12.0 statistical package (SPSS Inc., Chicago, IL, USA) and MedCalc 9.6.4.0 (MedCalc Software, Mariakerke, Belgium). The categorical variables are presented as percentages, and the continuous variables are presented as mean ± standard deviation if normally distributed or median (interquartile range) if not normally distributed. Statistical significance for intergroup differences was assessed by chi-square or
Potential predictors of 6-month mortality are shown in Table 2. Patients who experienced 6-month mortality had higher glucose, C-reactive protein and leptin level, lower GCS score, and more frequently showed unreactive pupils, CT classification 5 or 6, abnormal cisterns, midline shift >5 mm and traumatic subarachnoid hemorrhage on initial CT scan, and need more mechanical ventilation. Multivariate logistic regression analysis showed that variables
2.5. End point
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Fig. 1. Graph documenting patients’ entry into the study from screening.
Table 2 The main baseline demographic, clinical, and radiologic characteristics associated with 6-month mortality for 142 head trauma children.
Number Sex (male/female) Age (month) GCS score on admission Pediatric trauma score on admission Injury severity score on admission Pupils unreactive on admission CT classification 5 or 6 Abnormal cisterns on initial CT scan Midline shift >5 mm on initial CT scan Traumatic SAH on initial CT scan Mechanical ventilation Intracranial surgery in 1st 24 h Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Heart rate (beats/min) Body temperature (◦ C) Respiratory rate (respirations/min) Blood glucose level (mmol/L) Plasma C-reactive protein level (mg/L) Plasma leptin level (ng/mL)
Nonsurvival Group
Survival Group
P value
26 (18.3%) 19/7 72 (60) 4 (2) 6 (3) 27 (6) 23 (88.5%) 22 (84.6%) 21 (80.8%) 19 (73.1%) 20 (76.9%) 26 (100.0%) 16 (61.5%) 3.2 ± 1.5 3.7 ± 1.7 98.5 ± 18.4 62.7 ± 13.6 107.0 ± 15.9 37.0 ± 0.7 23.6 ± 3.5 11.6 ± 4.0 7.8 ± 2.7 20.1 ± 5.1
116 (81.7%) 77/39 60 (65) 6 (3) 5 (4) 27 (3) 33 (28.5%) 46 (39.7%) 38 (32.8%) 45 (38.8%) 55 (47.4%) 95 (81.9%) 56 (48.3%) 3.0 ± 1.3 3.4 ± 1.4 95.3 ± 20.3 60.2 ± 11.5 108.0 ± 22.3 37.1 ± 0.8 25.0 ± 4.4 9.2 ± 4.4 6.5 ± 2.6 10.9 ± 6.3
0.510 0.310 <0.001 0.380 0.856 <0.001 <0.001 <0.001 0.001 0.006 0.014 0.221 0.165 0.242 0.459 0.334 0.823 0.696 0.130 0.012 0.026 <0.001
The categorical variables are presented as counts (percentage), and the continuous variables are presented as mean ± standard deviation if normally distributed or median (interquartile range) if not normally distributed. Statistical significance for intergroup differences was assessed by chi-square or Fisher exact test for categorical variables, and by Student’s t or Mann–Whitney U test for continuous variables. GCS, Glasgow Coma scale; CT, computerized tomography; SAH, subarachnoid hemorrhage.
Table 3 Receiver operating characteristic curve analysis of factors predicting 6-month mortality for 142 head trauma children.
Criterion Area under curve (95% CI) Sensitivity (95% CI) Specificity (95% CI) Positive likelihood ratio (95% CI) Negative likelihood ratio (95% CI) Positive predictive value (95% CI) Negative predictive value (95% CI) P value GCS indicates Glasgow Coma scale; CI, confidence interval.
Leptin
GCS score
>14.4 ng/mL 0.859 (0.791–0.912) 96.2 (80.3–99.4) 65.6 (56.1–74.1) 2.79 (2.4–3.2) 0.059 (0.008–0.4) 38.5 (26.7–51.4) 98.7 (92.9–99.8) Reference
<5 0.905 (0.845–0.948) 92.3 (74.8–98.8) 70.7 (61.5–78.8) 3.15 (2.7–3.7) 0.11 (0.03–0.4) 41.4 (28.6–55.1) 97.6 (91.6–99.6) 0.303
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4. Discussion
Fig. 2. Graph showing receiver operating characteristic curve analysis of plasma leptin level for 6-month mortality.
independently related to 6-month mortality were plasma leptin level (OR 1.225, 95% CI 1.102–1.519, P = 0.006) and GCS score (OR 0.374, 95% CI 0.232–0.671, P = 0.001). ROC curves identified cutoff points for plasma leptin level on admission as the value that better predicted 6-month mortality (Fig. 2). The predictive value of the leptin concentration was thus similar to that of GCS score (Table 3). 3.3. Impact of plasma leptin level on the unfavorable outcome at 6 months Potential predictors of 6-month unfavorable outcome are shown in Table 4. Patients who experienced 6-month unfavorable outcome had higher glucose, C-reactive protein and leptin level, lower GCS score, and more frequently showed unreactive pupils, CT classification 5 or 6, abnormal cisterns, midline shift >5 mm and traumatic subarachnoid hemorrhage on initial CT scan, and need more mechanical ventilation. Multivariate logistic regression analysis showed that variables independently related to 6-month unfavorable outcome were plasma leptin level (OR 1.340, 95% CI 1.162–1.678, P = 0.002) and GCS score (OR 0.331, 95% CI 0.204–0.629, P = 0.001). ROC curves identified cutoff points for plasma leptin level on admission as the value that better predicted 6-month unfavorable outcome (Fig. 3). The predictive value of the leptin concentration was thus similar to that of GCS score (Table 5).
Fig. 3. Graph showing receiver operating characteristic curve analysis of plasma leptin level for 6-month functional outcome.
This study was conducted to determine if leptin is increased in the circulation of children with TBI and whether this increment correlates with 6-month clinical outcomes in these pediatric patients. The admission plasma leptin levels were indeed significantly increased in all patients compared with healthy subjects. Furthermore, an admission plasma leptin level was identified as a reliable and independent marker to predict patients at risk of 6month poor clinical outcomes. Importantly, the prognostic value of leptin was similar to that of GCS score for 6-month clinical outcomes, substantiating its potential as a new prognostic biomarker. Originally thought to be a satiety factor, leptin is a pleiotropic molecule [25]. Since its cloning in 1994, leptin’s role in regulating inflammatory response has become increasing evident. Both the structure of leptin and that of its receptor suggest that leptin might be classified as a cytokine [14]. The secondary structure of leptin has similarities to the long-chain helical cytokines family, which includes interleukin-6, interleukin-1, leukemia inhibitory factor, and ciliary neurotrophic factor [36]. Leptin receptors are encoded by the diabetes gene and homologous to the gp-130 signaltransducing subunit of the interleukin-6-type cytokine receptors [7]. Indeed, leptin regulates several cytokine secretion patterns. It has been shown that different inflammatory stimuli, including interleukin-1, interleukin-6, tumor necrosis factor-alpha or lipopolysaccharide, regulate leptin mRNA expression as well as circulating leptin levels [13,24,27]. Furthermore, leptin is produced by inflammatory-regulatory cells [26]. Conversely, leptin enhances the production of C-reactive protein, tumor necrosis-alpha and interkeulin-6, suggesting that leptin expression could participate in the inflammatory process [10,17,35]. Actually, the increase of leptin production that occurs during infection and inflammation strongly suggests that leptin is a part of the cytokines loop [4,12]. In central nervous system, brain cortex leptin mRNA expression and serum leptin level are up-regulated in ischemic mouse brain, as well as in rat brain with traumatic brain injury [8,32,33]. Concerning experimental autoimmune encephalomyelitis, it has been shown that leptin-deficient ob/ob mice are resistance to the development of this model of multiple sclerosis. This resistance is abolished by the administration of leptin, which is accompanied by a switch from a TH2 to TH1 pattern of cytokine release [22]. In addition, it has been noticed that the onset of multiple sclerosis is preceded by an increase of circulating leptin [21]. Noteworthy, it has been shown that leptin is expressed by both macrophages and T-cells infiltrated into the central nervous system during autoimmune encephalomyelitis [26]. This interesting report indicates that leptin is produced by immune cells during acute autoimmune encephalomyelitis. Recently, it is evidenced that administration of leptin increased levels of interleukin-1 beta in the hypothalamus in the rat [18]. It is suggested that leptin could participate in the development of central nervous system-inflammatory diseases. Brain injury is known to increase expression of several inflammatory cytokines, such as interleukin-1, interleukin-6, and tumor necrosis factor-alpha [3,9], which are capable of inducing leptin gene expression [13,24,27]. In intracerebral hemorrhage, plasma leptin level was found to be related to C-reactive protein level [11]. These findings suggested leptin may contribute to the inflammatory process of brain injury. This study found increased plasma leptin level in children after TBI in association with a worse clinical outcome. To our knowledge, this is the first time that the relationship of plasma leptin level with outcome has been investigated soon after TBI. In this study, plasma leptin level was highly associated with 6-month clinical outcome in children with TBI. Therefore, the determination of leptin in the plasma of pediatric patients on admission provides the ability to distinguish between patients with 6-month good and bad outcome.
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Table 4 The main baseline demographic, clinical, and radiologic characteristics associated with 6-month unfavorable outcome for 142 head trauma children.
Number Sex (male/female) Age (month) GCS score on admission Pediatric trauma score on admission Injury severity score on admission Pupils unreactive on admission CT classification 5 or 6 Abnormal cisterns on initial CT scan Midline shift > 5 mm on initial CT scan Traumatic SAH on initial CT scan Mechanical ventilation Intracranial surgery in 1st 24 h Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Heart rate (beats/min) Body temperature (◦ C) Respiratory rate (respirations/min) Blood glucose level (mmol/L) Plasma C-reactive protein level (mg/L) Plasma leptin level (ng/mL)
Unfavorable outcome
Favorable outcome
P value
42 (29.6%) 29/13 72 (85) 4 (1) 5 (3) 27 (6) 30 (71.4%) 31 (73.8%) 29 (69.0%) 27 (64.3%) 30 (71.4%) 42 (100.0%) 25 (59.5%) 3.4 ± 1.6 3.8 ± 1.7 98.3 ± 20.7 61.7 ± 13.2 110.9 ± 17.1 37.0 ± 0.7 24.0 ± 4.4 10.8 ± 4.0 7.9 ± 2.9 19.6 ± 4.4
100 (70.4%) 67/33 72 (60) 7 (2) 6 (4) 27 (3) 26 (26.0%) 37 (37.0%) 30 (30.0%) 37 (37.0%) 45 (45.0%) 79 (79.0%) 47 (47.0%) 3.0 ± 1.3 3.4 ± 1.5 94.9 ± 19.7 60.2 ± 11.3 106.5 ± 2.7 37.1 ± 0.8 25 ± 4.2 9.1 ± 4.5 6.3 ± 2.4 9.6 ± 4.7
0.812 0.588 <0.001 0.292 0.552 <0.001 <0.001 <0.001 0.003 0.004 0.001 0.173 0.103 0.182 0.351 0.495 0.266 0.613 0.191 0.037 0.002 <0.001
The categorical variables are presented as counts (percentage), and the continuous variables are presented as mean ± standard deviation if normally distributed or median (interquartile range) if not normally distributed. Statistical significance for intergroup differences was assessed by chi-square or Fisher exact test for categorical variables, and by Student’s t or Mann–Whitney U test for continuous variables. GCS, Glasgow Coma scale; CT, computerized tomography; SAH, subarachnoid hemorrhage.
Table 5 Receiver operating characteristic curve analysis of factors predicting 6-month unfavorable outcome for 142 head trauma children.
Criterion Area under curve (95% CI) Sensitivity (95% CI) Specificity (95% CI) Positive likelihood ratio (95% CI) Negative likelihood ratio (95% CI) Positive predictive value (95% CI) Negative predictive value (95% CI) P value
Leptin
GCS score
>14.1 ng/mL 0.891 (0.827–0.937) 92.9 (80.5–98.4) 72.0 (62.1–80.5) 3.32 (2.9–3.8) 0.099 (0.03–0.3) 58.2 (45.5–70.1) 96.0 (88.7–99.1) Reference
<5 0.936 (0.883–0.970) 90.5 (77.4–97.3) 80.0 (70.8–87.3) 4.52 (3.9–5.2) 0.12 (0.04–0.3) 65.5 (51.9–77.5) 95.2 (88.2–98.7) 0.192
GCS, Glasgow Coma scale; CI, confidence interval.
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