Cerebrospinal Fluid Total Iron and Fe2+ Strongly Associated with The Incidence of Hydrocephalus in Intracerebral Hemorrhage Patients with Intraventricular Extension

doi:10.21203/rs.3.rs-4073754/v1

Background

Hydrocephalus (HCP) is a known complication of spontaneous intracerebral hemorrhage (ICH) extension into the ventricles (IVH) and is associated with poorer patient outcomes. The mechanisms in HCP development are not yet clear. Biomarkers in CSF could predict the risk of shunt-dependent hydrocephalus, to improve clinical outcomes in these patients, and could be more reliable than CT-scan evaluation.

Aim

This study aimed to test the reliability of measuring Fe²⁺, Fe³⁺, total iron concentration, and high mobility group box 1 (HMGB1) protein levels as CSF biomarkers to predict the risk of shunt-dependent hydrocephalus in ICH + IVH patients. We compared marker reliability with CT-scan mGraeb score to predict hydrocephalus.

Methods

All adult ICH + IVH patients with hydrocephalus admitted to Siloam Hospitals Lippo Village between March 2021 until December 2023 were included in this cohort study. The patient's clinical and radiological characteristics were retrieved, and CSF samples were obtained during EVD placement. Statistical analyses were performed to reveal potential clinical, radiological, and biomarker values as predictors of shunt-dependent hydrocephalus in ICH + IVH patients.

Results

Thirty-six ICH + IVH + HCP patients, including 16 females (44.4%) and 20 males (55.6%) were included in this study, with mean age of 60.75 years. Seventeen patients (47.2%) were diagnosed with shunt-dependent hydrocephalus. Logistic regression analyses revealed that increased total Fe (p = 0.048) and Fe2+ (0.045) were significantly associated with the incidence of shunt-dependent hydrocephalus. Receiver operating curves (ROC) revealed that the AUCs from total iron (0.716), Fe2+ (0.758), Fe3+ (0.651), and HMGB1 (0.722) are higher than the AUC of mGraeb score (0.528).

Conclusion

Based on the results from logistic regression analyses and constructed ROCs, CSF total iron and Fe²⁺ are reliable prognosticators of shunt-dependent hydrocephalus in ICH + IVH + HCP patients. These biomarkers are more sensitive and specific in predicting hydrocephalus compared to mGraeb score.

Non-traumatic intracerebral hemorrhage (ICH) is a stroke subtype that has high rates of mortality and morbidity.^1–3 Hydrocephalus, as a complication of ICH with intraventricular extension (IVH), has been proven to worsen the prognosis of ICH patients.⁴ A recent meta-analysis concluded hydrocephalus is an independent factor that increases the risks of mortality and morbidity in ICH patients. ^5,6

Timely diagnosis and treatment are key to the optimalization of ICH patients’ management. Obstructive hydrocephalus due to blood clots in the ventricle system is easily diagnosed using CT-scan,⁶ but delayed hydrocephalus is more difficult to predict; thus, many studies had been done to develop predictors of shunt-dependent hydrocephalus in ICH + IVH patients. ^7–11

In patients with obstructive hydrocephalus, who had been given temporary CSF diversion with an external ventricular drainage (EVD), not all of them will progress to shunt-dependent hydrocephalus.^12–15 As the hematoma that blocked the CSF flow lyse, other mechanisms have been proposed that could cause shunt-dependent hydrocephalus, such as ferroptosis, inflammation of the ventricular system, and other unknown mechanisms.^16–18 Based on this theory, we hypothesize that the amount of intraventricular hematoma, levels of CSF iron, and inflammatory proteins will be significantly associated with the risk of shunt-dependent hydrocephalus in ICH + IVH + HCP patients.

Based on this hypothesis, this study aims to test whether specific clinical characteristics and/or CSF biomarkers, including total iron, Fe²⁺, Fe³⁺, and HMGB1, could be used as reliable predictors of shunt-dependent hydrocephalus. If the occurrence of shunt-dependent hydrocephalus can be predicted prior to the EVD placement, then ICH + IVH + HCP patients can undergo shunt placement surgery directly instead of having an initial EVD placed. This will improve the efficiency of ICH patient management, reduce surgical burden, and minimize the risk of infection.

Patient Identification and Data Collection

The Ethical Review Board of the Faculty of Medicine, Universitas Pelita Harapan/Siloam Hospital (Ethical Approval No.147/K-LKJ/ETIK/IV/2023) has granted ethical approval for this study. All patients above 18 years old who were diagnosed with ICH + IVH + HCP, with onset under 24 hours were included in this cohort study with consecutive, non-probability sampling. Patients with underlying causes of hemorrhage (aneurysm, AVMs, etc), a history of systemic or autoimmune diseases, or previous nervous system inflammation were excluded. Data on the patient’s demographic, clinical, and radiological characteristics were collected.

Determination of Radiological Data

All authors independently reviewed anonymized CT scan data to calculate the ICH and modified Graeb (mGraeb) scores. These variables were calculated as previously described.^19,20 a

Cerebrospinal Fluid Collection

All patients underwent CSF diversion by external ventricular drainage (EVD) when diagnosed with hydrocephalus. 5 ml of CSF sample was collected from every patient during EVD placement.

Determination of Shunt Dependency

After five days, rapid shunt dependency test was performed as previously described by Bertuccio et al.²¹ In brief, patients who experienced clinical signs of increased intracranial pressure, confirmed by follow-up head CT scan when the EVD was clamped, were diagnosed with shunt-dependent hydrocephalus, and had conversion to ventriculoperitoneal (VP) shunt placement.

Detection of CSF Total Iron, Fe2+, and Fe3+

The total iron, Fe2+, and Fe3 + presences in the CSF samples were detected by using an iron colorimetric assay kit ABCAM ab83366 (Cambridge Biomedical Campus, Cambridge, UK). Briefly, CSF sample was added to the wells. Fe²⁺ assay was performed using assay buffer only, while total iron and Fe³⁺ measurements were performed by adding the iron reducer. Wells were incubated for 30 min at 37°C, Iron probe was added and incubated for 60 min at 37°C, and samples were analyze with microplate reader.

Detection of CSF HMGB1 protein levels

Detection of HMGB1 protein was performed using HMG1 enzyme linked immunosorbent assay Elisa Kit from MBS721722 (San Diego, CA, USA). After plotting and adjusting the sample values into the standard curve, calculation of HMGB1 concentration was performed and presented in pg/ml.

Data Analysis

Baseline characteristics were presented using mean (standard deviation) or median (min, max); categorical data were presented using frequency. Logistic regression was performed to evaluate the association between the incidence of shunt-dependent hydrocephalus and each of the variables. In addition to the p-values, the crude odds ratio and 95% confidence interval were presented. Receiver operating characteristic (ROC) curves were constructed to measure the area under the curves (AUC) and their 95% confidence intervals. All statistical tests were conducted using a 5% significance level.

Study Sample

Thirty-six ICH + IVH + HCP patients, including 16 females (44.4%) and 20 males (55.6%) were included in this study. The mean age of patients was 60.75 years; the median admission GCS was 10, with 26 patients (72.2%) of GCS 5–12 and 10 patients (27.8%) of GCS 13–15.

The ICH volume among these patients ranged from 2.75–44.77 ml, with median volume 10.62 ml. Ten patients (27.8%) had cumulative ICH score 1, 4 (11.1%) had ICH score 2, and 22 (61.1%) had ICH score 3. The most common bleeding location was the thalamus (n = 24; 66.7%), basal ganglia (n = 7; 19.4%), cerebellum (n = 3; 8.3%), and occipital lobe (n = 2; 5.6%). The mGraeb scores ranged from 2–27, with the median mGraeb score of 15.

Based on EVD challenge test, 17 patients (47.2%) were diagnosed with shunt-dependent hydrocephalus, while 19 patients (52.8%) were not. (Table 1).

Table 1: Patient characteristics

Characteristics

n=36

Gender, frequency (%)

Male

Female

16 (44.4)

20 (55.6)

Age (years), mean (SD)

60.75 (12.3)

GCS, median (min, max)

10 (5, 15)

GCS criteria for ICH score, frequency (%)

3 – 4

5 – 12

13 – 15

0 (0)

26 (72.2)

10 (27.8)

ICH volume (ml), median (min, max)

10.62 (2.75, 44.77)

ICH score, frequency (%)

0

1

0 (0)

10 (27.8)

2

3

4

5

4 (11.1)

22 (61.1)

0 (0)

ICH location, frequency (%)

Thalamus

Basal Ganglia

Cerebellum

Occipital

24 (66.7)

7 (19.4)

3 (8.3)

2 (5.6)

mGraeb score, median (min, max)

Shunt dependency, frequency (%)

Shunt-dependent hydrocephalus

Temporary hydrocephalus (EVD only)

15 (2, 27)

17 (47.2)

19 (52.8)

Logistic Regression Analyses of The Association of Shunt-Dependent Hydrocephalus With Clinical And Radiological Characteristics.

Clinical characteristics evaluated included age, gender, and admission GCS; the radiological characteristics evaluated included ICH location, ICH volume, ICH score, and mGraeb score. Four CSF biomarkers were tested in this study, including total iron, Fe²⁺, Fe³⁺, and HMGB1.

The results of the logistic regression analyses revealed that the associations of shunt-dependent hydrocephalus in ICH + IVH + HCP patients with age (p = 0.238), gender (p = 1.000), GCS (p = 0.146), ICH location (p = 0.164), ICH volume (p = 0.224), ICH score (2 vs 1 and 3 vs 1) (p = 0.256 and p = 0.600), mGraeb score (0.894), HMGB1 (0.493), and Fe³⁺ (p = 0.180) are not statistically significant; meanwhile, total Fe (p = 0.048) and Fe²⁺ (0.045) are significantly associated with shunt-dependent hydrocephalus in ICH + IVH + HCP patients (Table 2).

Table 2

Univariate Association analysis for Hydrocephalus incidence
Effect	β	SE(β)	Wald	Crude-OR (95% CI)	p-value
Age	0.034	0.029	1.39	1.034 (0.979, 1.098)	0.238
Gender (male vs female)	0.000	0.671	0.00	1.000 (0.265, 3.770)	1.000
GCS (5–12 vs 13–15)	1.158	0.796	2.11	3.182 (0.710, 17.469)	0.146
ICH location (Thalamus vs others)	-1.030	0.739	1.94	0.357 (0.077, 1.463)	0.164
ICH volume	0.040	0.033	1.48	1.041 (0.979, 1.116)	0.224
ICH score (2 vs 1)	1.504	1.323	1.29	4.500 (0.409, 111.103)	0.256
ICH score (3 vs 1)	0.406	0.774	0.28	1.500 (0.333, 7.313)	0.600
mGraeb score	0.007	0.053	0.02	1.007 (0.906, 1.121)	0.894
HMGB-1	-0.068	0.997	0.47	0.934 (0.741, 1.129)	0.493
Total Fe	0.006	0.003	3.91	1.006 (1.001, 1.014)	0.048*
Fe²⁺	0.012	0.006	4.00	1.012 (1.002, 1.027)	0.045*
Fe³⁺	0.011	0.008	1.80	1.011 (1.000, 1.027)	0.180
β = regression coefficient; SE = standard error; OR = odds ratio; CI = confidence interval
HMGB-1 was centered at 1
Fe²⁺, Fe³⁺ and Total Fe were centered at 100
Age was centered at 40
ICH volume was centered at 30
mGraeb score was centered at 10

Receiver Operating Curves of MGraeb Score, Total Iron, Fe ²⁺, Fe³⁺, And HMGB1 for Prediction of Shunt-Dependent Hydrocephalus

To compare the reliability between mGraeb score and the evaluated biomarkers in predicting the risk of shunt-dependent hydrocephalus in ICH + IVH + HCP patients, receiver operating curves (ROC) were constructed. The plotted ROC revealed that the AUCs from total iron (0.716), Fe²⁺ (0.758), Fe³⁺ (0.651), and HMGB1 (0.722) are higher than the AUC of mGraeb score (0.528) (Fig. 1A-D).

This study evaluated potential clinical, radiological, and biological variables that might be significantly associated with the incidence of shunt-dependent hydrocephalus ICH + IVH + HCP patients. Clinical and radiological characteristics, such as age, admission GCS, and ICH volume, have been associated with the incidence of hydrocephalus in human or animal models^7,8,22, and this study evaluated age, gender, admission GCS, ICH volume, and ICH score as potential variables that might be related with the incidence of shunt-dependent hydrocephalus. As ICH in infratentorial regions is more likely to cause hydrocephalus,⁹ this study also included ICH location as a potential variable. All these markers were compared to the mGraeb score, which is a regularly used indicator of intraventricular hematoma.

In shunt-dependent hydrocephalus, the hemolysis of intraventricular hematoma releases iron molecules, causing iron-mediated inflammation that leads to the scarring or fibrosis of arachnoid granulation that impairs CSF absorption. Based on this theory, it is logical to assume that the increase in IVH severity, iron levels, and inflammatory markers would represent higher risks of shunt-dependent hydrocephalus. Logistic regression analyses revealed that total iron and Fe²⁺ are significantly associated with shunt-dependent hydrocephalus. This finding supports previous studies that suggested ferroptosis as a good indicator of post hemorrhagic hydrocephalus in animal models.²³ It is interesting to notice that not all forms of iron molecules are elevated in ICH + IVH + HCP patients who were later identified as having shunt-dependent hydrocephalus. One possible explanation for this finding is that the available biological mechanisms convert Fe³⁺ to Fe^2 + 24, and so Fe²⁺ levels are higher in these patients.

It is interesting to see that HMGB1 level was not significantly elevated in our patients. As inflammation is one of the suggested mechanisms in causing shunt-dependent hydrocephalus, it was hoped that pre-inflammatory marker HMGB-1 would be elevated. Perhaps inflammation was not as important as ferroptosis in causing shunt-dependent hydrocephalus, or perhaps a non-HMGB1-mediated inflammatory pathway is involved.

Knowing that total iron and F²⁺, the products of hemolysis, are significantly associated with shunt-dependent hydrocephalus, it is safe to assume IVH severity should significantly affect hydrocephalus incidence as well. The mGraeb score is a semiquantitative IVH severity scoring system that is based on the amount of intraventricular hematoma; thus, it is logical to compare the AUCs between the mGraeb score and CSF biomarkers. Based on the plotted ROC, it is clearly visible that the AUC of all CSF biomarkers, including total iron, Fe²⁺, Fe³⁺, and HMGB1 are higher than mGraeb score. This suggests the potential use of these CSF biomarkers as more accurate predictors of shunt-dependent hydrocephalus in ICH + IVH + HCP patients.

This study showed that the amount of CSF total iron and Fe²⁺ are significantly associated with the development of shunt-dependent hydrocephalus and is more sensitive and specific compared to radiological evaluation using CT scan mGraeb score or HMGB-1 inflammation marker. This could be used as consideration when deciding to perform permanent CSF shunting or continuing temporary diversion. This would be beneficial to the patient, to avoid repetitive temporary CSF diversion and its associated risks, and perhaps to affect the initial decision whether to perform temporary or permanent CSF diversion in these patients. The fact that total iron and Fe²⁺ were significantly associated with hydrocephalus could also mark them as possible therapeutic targets in the prevention of shunt-dependent hydrocephalus.

The sample size of this study was small, as it was conducted in the midst of COVID-19 era. However, our results show good statistical power. Our insistence in evaluating only spontaneous ICH with no underlying pathologies also sets us apart from similar studies evaluating aneurysmal subarachnoid hemorrhage causing hydrocephalus, which may have a different mechanism of hydrocephalus development.

In the future, other iron-related biomarkers could be checked, including ferritin, transferrin, and radical oxygen species. Other inflammatory markers may be examined as well, such as DAMP, RAGE, or NF-kB.

AUTHORSHIP REQUIREMENTS

This manuscript complies with all instructions to authors.

Author contributions:

Petra Octavian Perdana Wahjoepramono : conception of the work. Patient recruitment, sample collection, data analysis, drafting the work. Corresponding author.

Aloysius Bagus Sasongko : co-conception of the work. Helping laboratory work. Data analysis, drafting the work.

Danny Halim : co-conception of the work. Designing laboratory protocol, data analysis, drafting the work.

Kurnia Wahyudi: co-conception of the work. Statistical analysis, results interpretation, discussion. Drafting the work.

Achmad Adam: co-conception of the work. Critical review and suggestion to study protocol. Review of the work, final approval.

Yeo Tseng Tsai: co-conception of the work. Critical review and suggestion to study protocol. Critical review of the results and discussion. Review of the work.

Eka Julianta Wahjoepramono: co-conception of the work. Critical review and suggestion to study protocol. Critical review of the results and discussion. Review of the work.

Julius July: co-conception of the work. Critical review and suggestion to study protocol. Critical review of the results and discussion. Review of the work.

Tri Hanggono Achmad: co-conception of the work. Critical review and suggestion to study protocol. Critical review of the results and discussion. Review of the work.

The final manuscript has been approved by all authors, and all authors agree to be held accountable for all aspects of the work.

This manuscript has not been published elsewhere and is not under consideration by another journal.

ETHICAL ISSUES

The Ethical Review Board of the Faculty of Medicine, Universitas Pelita Harapan/Siloam Hospital (Ethical Approval No.147/K-LKJ/ETIK/IV/2023) has granted ethical approval for this study.

INFORMED CONSENT

Informed consent was obtained from all individual participants included in the study.

COMPETING INTERESTS

The Authors have no competing interests in writing or publishing this paper.

FUNDINGS

The Authors did not receive any funding in writing or publishing this paper.

REPORTING CHECKLIST

The STROBE checklist has been completed and included at the end of the manuscript.

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STROBEStatement.docx

Cerebrospinal Fluid Total Iron and Fe2+ Strongly Associated with The Incidence of Hydrocephalus in Intracerebral Hemorrhage Patients with Intraventricular Extension

Status:

Version 1

Abstract

Background

Aim

Methods

Results

Conclusion

Figures

Introduction

Methods

Patient Identification and Data Collection

Determination of Radiological Data

Cerebrospinal Fluid Collection

Determination of Shunt Dependency

Detection of CSF Total Iron, Fe2+, and Fe3+

Detection of CSF HMGB1 protein levels

Data Analysis

Results

Study Sample

Discussion

Conclusion

Declarations

References

Supplementary Files

Status:

Version 1