Evaluation of Serum NLRC4 as a Potential Prognostic Biochemical Marker in Humans with Severe Traumatic Brain Injury: A Prospective Cohort Study

Figures & data

Table 1 Baseline Characteristics of Head Trauma Patients and Factors in Relation to Serum NLRC4 Levels After Head Trauma

	All Patients	ρ	P value
Gender (male/female)	74/66	−0.084	0.322
Age (y)	38.8±18.0	0.035	0.679
Body mass index (kg/m^2)	24.0±3.3	0.106	0.214
Current cigarette smoking	44 (31.4%)	0.071	0.406
Current alcohol drinking	45 (32.1%)	0.033	0.698
Hypertension	12 (8.6%)	0.125	0.141
Diabetes mellitus	8 (5.7%)	−0.065	0.447
Hyperlipidemia	19 (13.6%)	0.028	0.746
Previous use of antihypertensive drugs	8 (5.7%)	0.053	0.538
Previous use of hypoglycemic drugs or insulin	5 (3.6%)	0.018	0.836
Previous use of statins	11 (7.9%)	0.077	0.365
Admission time since trauma (h)	4.4 (1.8–6.5)	−0.107	0.210
Blood-collection time since trauma (h)	5.4 (2.8–8.1)	−0.118	0.165
Traumatic causes (automobile/motorcycle: fall/jump: others)	73/56/11	0.105	0.219
Glasgow coma scale scores	5 (4–6)	−0.608	<0.001
Rotterdam scores	4 (4–5)	0.589	<0.001
Unreactive pupils	88 (62.9%)	0.256	0.002
Surgery in the first 24 hours	80 (57.1%)	−0.043	0.612
Epileptic seizure	20 (14.3%)	−0.011	0.896
Systolic arterial pressure (mmHg)	123.4±25.2	−0.027	0.748
Diastolic arterial pressure (mmHg)	74.6±16.2	−0.008	0.921
Mean arterial pressure (mmHg)	91.1±18.1	−0.015	0.864
Epidural hematoma	67 (47.9%)	0.055	0.519
Subdural hematoma	74 (52.9%)	0.089	0.298
Subarachnoid hemorrhage	88 (62.9%)	0.102	0.232
Intraventricular hemorrhage	14 (10.0%)	0.027	0.756
Intracerebral hematoma	75 (53.6%)	0.077	0.368
Brain contusion	77 (55.0%)	0.119	0.162
Pneumocephalus	58 (41.4%)	−0.064	0.453
Abnormal cisterns	101 (72.1%)	0.208	0.014
Midline shift > 5 mm	88 (62.9%)	0.230	0.006
Serum C-reactive protein levels (mg/l)	14.6 (10.2–17.9)	0.420	<0.001
Blood glucose levels (mmol/l)	9.9 (8.3–11.5)	0.382	<0.001
Blood leucocyte count (×10^9/l)	7.1 (5.0–9.8)	0.240	0.004

Notes: Data were summarized as mean ± standard deviation, median (25th-75th percentiles) or count (percentage) as appropriate. Bivariate correlations were analyzed using Spearman test and correlations were reported as ρ values.

Figure 1 Serum NLRC4 levels after severe traumatic brain injury. Serum NLRC4 levels were substantially elevated after severe traumatic brain injury, in comparison to controls (P<0.001).

Figure 2 Correlative analysis of serum NLRC4 levels following severe traumatic brain injury. (A) Correlation of serum NLRC4 levels with Glasgow coma scale scores after severe traumatic brain injury. Serum NLRC4 levels were significantly correlated with Glasgow coma scale scores after severe traumatic brain injury (P<0.001). (B) Correlation of serum NLRC4 levels with Rotterdam computed tomography scores after severe traumatic brain injury. Serum NLRC4 levels were significantly correlated with Rotterdam computed tomography scores after severe traumatic brain injury (P<0.001). (C) Correlation of serum NLRC4 levels with serum C-reactive protein levels after severe traumatic brain injury. Serum NLRC4 levels were significantly correlated with serum C-reactive protein levels after severe traumatic brain injury (P<0.001).

Abbreviations: GCS, Glasgow coma scale; CT, computed tomography; CRP, C-reactive protein.

Figure 3 Serum NLRC4 levels among severe traumatic brain injury subgroups according to clinical and radiological severity scales. (A) Serum NLRC4 levels among subgroups with different Glasgow outcome scale scores after severe traumatic brain injury. Serum NLRC4 levels were significantly decreased in order of Glasgow outcome scale scores from 3 to 8 (P<0.001). (B) Serum NLRC4 levels among subgroups with different Rotterdam computerized tomography scores after severe traumatic brain injury. Serum NLRC4 levels were significantly increased in order of Rotterdam computerized tomography scores from 3 to 6 (P<0.001).

Abbreviations: GCS, Glasgow coma scale; CT, computed tomography.

Table 2 Factors in Relation to 180-Day Mortality After Head Trauma

	Non-Survivors	Survivors	P value
Gender (male/female)	19/14	55/52	0.535
Age (y)	43.7±18.0	37.2±17.8	0.070
Body mass index (kg/m^2)	24.2±3.2	23.9±3.4	0.743
Current cigarette smoking	14 (42.4%)	30 (28.0%)	0.120
Current alcohol drinking	16 (48.5%)	29 (27.1%)	0.021
Hypertension	2 (6.1%)	10 (9.4%)	0.732
Diabetes mellitus	3 (9.1%)	5 (4.7%)	0.392
Hyperlipidemia	5 (15.2%)	14 (13.0%)	0.762
Previous use of antihypertensive drugs	1 (3.0%)	7 (6.5%)	0.680
Previous use of hypoglycemic drugs or insulin	2 (6.1%)	3 (2.8%)	0.337
Previous use of statins	3 (9.1%)	8 (7.5%)	0.721
Admission time since trauma (h)	3.6 (2.1–6.0)	4.6 (1.8–6.5)	0.700
Blood-collection time since trauma (h)	4.6 (2.7–7.6)	5.7 (2.8–8.3)	0.551
Traumatic causes			0.600
Automobile/motorcycle	16	57
Fall/jump	13	43
Others	4	7
Glasgow coma scale scores	4 (3–4)	5 (5–6)	<0.001
Rotterdam scores	5 (5–6)	4 (4–5)	<0.001
Unreactive pupils	28 (84.9%)	60 (56.1%)	0.003
Surgery in the first 24 hours	22 (66.7%)	58 (54.2%)	0.206
Epileptic seizure	6 (18.2%)	14 (13.1%)	0.569
Systolic arterial pressure (mmHg)	129.1±23.8	121.6±25.5	0.136
Diastolic arterial pressure (mmHg)	77.6±16.3	73.7±16.1	0.229
Mean arterial pressure (mmHg)	95.9±16.7	89.6±18.4	0.081
Epidural hematoma	23 (69.7%)	44 (41.1%)	0.004
Subdural hematoma	23 (69.7%)	51 (47.7%)	0.027
Subarachnoid hemorrhage	25 (75.8%)	63 (58.9%)	0.079
Intraventricular hemorrhage	5 (15.2%)	9 (8.4%)	0.318
Intracerebral hematoma	20 (60.6%)	55 (51.4%)	0.354
Brain contusion	20 (60.6%)	57 (53.3%)	0.459
Pneumocephalus	14 (42.4%)	44 (41.1%)	0.894
Abnormal cisterns	31 (93.9%)	70 (65.4%)	0.001
Midline shift > 5 mm	27 (81.8%)	61 (57.0%)	0.010
Serum C-reactive protein levels (mg/l)	17.5 (15.7–21.8)	12.6 (9.6–17.0)	<0.001
Blood glucose levels (mmol/l)	11.4 (10.1–13.9)	9.7 (7.9–10.9)	0.001
Blood leucocyte count (×10^9/l)	12.0 (4.5–13.8)	6.3 (5.1–8.2)	0.001
Serum NLRC4 levels (ng/mL)	1.3 (0.8–1.5)	0.6 (0.3–1.1)	<0.001

Notes: Data were presented as mean ± standard deviation, median (25th-75th percentiles) or count (percentage) and were compared by the t-test, Mann–Whitney U-test, Pearson chi-square test or Fisher’s exact test.

Figure 4 Serum NLRC4 levels with respect to discriminatory ability for risk of 180-day death after severe traumatic brain injury. (A) Predictive value of serum NLRC4 levels for 180-day death after severe traumatic brain injury. Serum NLRC4 levels efficiently distinguished patients at risk of 180-day death following severe traumatic brain injury (P<0.001). (B) Differences in 180-day death predictive ability between serum NLRC4 levels and other variables among severe traumatic brain injury patients. Combination of serum NLRC4 levels, Glasgow coma scale scores and Rotterdam computerized tomography scores had insignificantly higher area under curve than Glasgow coma scale scores (P>0.05), while displayed substantially higher area under curve than Rotterdam computerized tomography scores (P<0.05).

Abbreviations: GCS, Glasgow coma scale; CT, computed tomography; AUC, area under curve; 95% CI, 95% confidence interval.

Table 3 Factors in Relation to 180-Day Overall Survival After Head Trauma

	Hazard Ratio	95% Confidence Interval	P value
Gender (male/female)	1.212	0.608–2.417	0.585
Age (y)	1.015	0.998–1.033	0.088
Body mass index (kg/m^2)	1.016	0.918–1.124	0.763
Current cigarette smoking	1.583	0.793–3.157	0.192
Current alcohol drinking	2.114	1.068–4.185	0.032
Hypertension	0.682	0.163–2.851	0.600
Diabetes mellitus	1.968	0.600–6.449	0.264
Hyperlipidemia	1.274	0.492–3.299	0.619
Previous use of antihypertensive drugs	0.465	0.064–3.403	0.451
Previous use of hypoglycemic drugs or insulin	1.928	0.461–8.062	0.369
Previous use of statins	1.299	0.396–4.258	0.665
Admission time since trauma (h)	0.972	0.873–1.081	0.596
Blood-collection time since trauma (h)	0.964	0.873–1.064	0.465
Traumatic causes
Automobile/motorcycle	Reference
Fall/jump	0.537	0.179–1.608	0.267
Others	0.588	0.192–1.804	0.353
Glasgow coma scale scores	0.359	0.250–0.516	<0.001
Rotterdam cores	3.663	2.352–5.706	<0.001
Unreactive pupils	3.719	1.436–9.636	0.007
Surgery in the first 24 hours	1.542	0.748–3.181	0.241
Epileptic seizure	1.515	0.625–3.670	0.358
Systolic arterial pressure (mmHg)	1.011	0.997–1.025	0.115
Diastolic arterial pressure (mmHg)	1.014	0.992–1.037	0.215
Mean arterial pressure (mmHg)	1.018	0.998–1.039	0.073
Epidural hematoma	2.788	1.326–5.861	0.007
Subdural hematoma	2.224	1.058–4.674	0.035
Subarachnoid hemorrhage	2.006	0.905–4.448	0.087
Intraventricular hemorrhage	1.712	0.661–4.436	0.268
Intracerebral hematoma	1.362	0.677–2.738	0.386
Brain contusion	1.281	0.637–2.575	0.487
Pneumocephalus	1.092	0.547–2.178	0.803
Abnormal cisterns	6.723	1.608–28.102	0.009
Midline shift > 5 mm	3.028	1.250–7.337	0.014
Serum C-reactive protein levels (mg/l)	1.189	1.108–1.275	<0.001
Blood glucose levels (mmol/l)	1.132	1.048–1.222	0.002
Blood leucocyte count (×10^9/l)	1.301	1.175–1.440	<0.001
Serum NLRC4 levels (ng/mL)	3.946	1.881–8.279	<0.001

Notes: Associations were shown as hazard ratios (95% confidence intervals) using univariate Cox’s proportional hazard regression model.

Figure 5 Difference in 180-day overall survival time according to serum NLRC4 levels after severe traumatic brain injury. Based on median value of serum NLRC4 levels (namely, 0.83 ng/mL), patients were dichotomized into two subgroups. Patients with serum NLRC4 levels > 0.83 ng/mL exhibited markedly shorter 180-day overall survival time than other remainders (P<0.01).

Table 4 Factors in Relation to Extended Glasgow Outcome Scale Scores at 180 Days After Head Trauma

	ρ	P value
Gender (male/female)	−0.099	0.244
Age (y)	−0.154	0.068
Body mass index (kg/m^2)	−0.144	0.090
Current cigarette smoking	−0.205	0.015
Current alcohol drinking	−0.092	0.280
Hypertension	−0.071	0.406
Diabetes mellitus	−0.133	0.117
Hyperlipidemia	0.017	0.844
Previous use of antihypertensive drugs	0.039	0.644
Previous use of hypoglycemic drugs or insulin	−0.134	0.115
Previous use of statins	−0.067	0.434
Admission time since trauma (h)	−0.041	0.629
Blood-collection time since trauma (h)	−0.012	0.886
Traumatic causes	−0.009	0.918
Glasgow coma scale scores	0.664	<0.001
Rotterdam scores	−0.624	<0.001
Unreactive pupils	−0.316	<0.001
Surgery in the first 24 hours	−0.162	0.056
Epileptic seizure	−0.106	0.213
Systolic arterial pressure (mmHg)	−0.138	0.104
Diastolic arterial pressure (mmHg)	−0.062	0.468
Mean arterial pressure (mmHg)	−0.140	0.098
Epidural hematoma	−0.331	<0.001
Subdural hematoma	−0.145	0.088
Subarachnoid hemorrhage	−0.117	0.169
Intraventricular hemorrhage	−0.010	0.908
Intracerebral hematoma	−0.130	0.127
Brain contusion	−0.229	0.007
Pneumocephalus	0.130	0.125
Abnormal cisterns	−0.258	0.002
Midline shift > 5 mm	−0.354	<0.001
Serum C-reactive protein levels (mg/l)	−0.379	<0.001
Blood glucose levels (mmol/l)	−0.388	<0.001
Blood leucocyte count (×10^9/l)	−0.166	0.050
Serum NLRC4 levels (ng/mL)	−0.507	<0.001

Note: Bivariate correlations were assessed by Spearman test.

Figure 6 Relationship between serum NLRC4 levels and 180-day extended Glasgow outcome scale scores after severe traumatic brain injury. (A) Correlation of serum NLRC4 levels with 180-day extended Glasgow outcome scale scores after severe traumatic brain injury. Serum NLRC4 levels were in close correlation with 180-day extended Glasgow outcome scale scores after severe traumatic brain injury (P<0.001). (B) Difference in serum NLRC4 levels among subgroups with different 180-day extended Glasgow outcome scale scores after severe traumatic brain injury. Serum NLRC4 levels were dramatically declined in order of 180-day extended Glasgow outcome scale scores from 1 to 8 (P<0.001).

Abbreviation: GOSE, Extended Glasgow outcome scale.

Table 5 Factors in Relation to 180-Day Poor Outcome After Head Trauma

	Poor Outcome	Good Outcome	P value
Gender (male/female)	38/29	36/37	0.381
Age (y)	39.2±17.3	38.3±18.7	0.774
Body mass index (kg/m^2)	24.5±3.5	23.6±3.1	0.114
Current cigarette smoking	26 (38.8%)	18 (24.7%)	0.072
Current alcohol drinking	23 (34.3%)	22 (30.1%)	0.596
Hypertension	7 (10.5%)	5 (6.9%)	0.447
Diabetes mellitus	6 (9.0%)	2 (2.7%)	0.152
Hyperlipidemia	9 (13.4%)	10 (13.7%)	0.963
Previous use of antihypertensive drugs	5 (7.5%)	3 (4.1%)	0.480
Previous use of hypoglycemic drugs or insulin	4 (6.0%)	1 (1.4%)	0.193
Previous use of statins	7 (10.5%)	4 (5.5%)	0.275
Admission time since trauma (h)	4.4 (2.1–7.1)	4.3 (1.6–6.2)	0.390
Blood-collection time since trauma (h)	5.3 (3.1–8.2)	5.5 (2.5–7.9)	0.657
Traumatic causes			0.226
Automobile/motorcycle	33	40
Fall/jump	26	30
Others	8	3
Glasgow coma scale scores	4 (4–5)	6 (5–7)	<0.001
Rotterdam scores	5 (4–6)	4 (4–4)	<0.001
Unreactive pupils	50 (74.6%)	38 (52.1%)	0.006
Surgery in the first 24 hours	42 (62.7%)	38 (52.1%)	0.204
Epileptic seizure	12 (17.9%)	8 (11.0%)	0.240
Systolic arterial pressure (mmHg)	125.3±26.8	121.6±23.7	0.376
Diastolic arterial pressure (mmHg)	74.8±18.0	74.4±14.5	0.887
Mean arterial pressure (mmHg)	92.2±19.6	90.1±16.8	0.491
Epidural hematoma	40 (59.7%)	27 (37.0%)	0.007
Subdural hematoma	39 (58.2%)	35 (48.0%)	0.224
Subarachnoid hemorrhage	46 (68.7%)	42 (57.5%)	0.174
Intraventricular hemorrhage	7 (10.5%)	7 (9.6%)	0.866
Intracerebral hematoma	41 (61.2%)	34 (46.6%)	0.083
Brain contusion	45 (67.2%)	32 (43.8%)	0.006
Pneumocephalus	23 (34.3%)	35 (48.0%)	0.102
Abnormal cisterns	57 (85.1%)	44 (60.3%)	0.001
Midline shift > 5 mm	52 (77.6%)	36 (49.3%)	0.001
Serum C-reactive protein levels (mg/l)	16.8 (12.7–21.0)	12.3 (9.2–16.7)	<0.001
Blood glucose levels (mmol/l)	11.0 (9.8–13.5)	9.3 (7.3–9.9)	<0.001
Blood leucocyte count (×10^9/l)	8.0 (4.5–12.1)	7.0 (5.1–8.3)	0.323
Serum NLRC4 levels (ng/mL)	1.2 (0.8–1.5)	0.5 (0.2–1.0)	<0.001

Notes: Data were presented as mean ± standard deviation, median (25th–75th percentiles) or count (percentage) and were compared by the t-test, Mann–Whitney U-test, Pearson chi-square test or Fisher’s exact test.

Figure 7 Serum NLRC4 levels concerning distinguishable capability for risk of 180-day poor outcome after severe traumatic brain injury. (A) Predictive ability of serum NLRC4 levels for 180-day poor prognosis after severe traumatic brain injury. Serum NLRC4 levels substantially distinguished patients with development of 180-day poor prognosis following severe traumatic brain injury (P<0.001). (B) Differences in 180-day prognostic predictive ability between serum NLRC4 levels and other parameters among severe traumatic brain injury patients. Serum NLRC4 levels combined with Glasgow coma scale scores and Rotterdam computerized tomography scores exhibited profoundly higher area under curve than Rotterdam computerized tomography scores and Glasgow coma scale scores alone (both P<0.05).

Abbreviations: GCS, Glasgow coma scale; CT, computed tomography; AUC, area under curve; 95% CI, 95% confidence interval.

Data Sharing Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.