Association of inflammatory cytokines expression in cerebrospinal fluid with the severity and prognosis of spontaneous intracerebral hemorrhage

doi:10.21203/rs.3.rs-3333381/v1

Objective

To investigate the potential diagnostic and prognostic implications of inflammatory cytokine levels in the cerebrospinal fluid (CSF) of Spontaneous intracerebral hemorrhage (SICH) patients upon their initial hospital admission.

Methods

Our cohort comprised 100 patients diagnosed with acute SICH, who presented to the Department of Neurosurgery. Additionally, 50 individuals, devoid of central nervous system (CNS) pathology and treated concomitantly at our facility, were enlisted as controls. CSF specimens, acquired upon hospital entry, underwent quantitative assessment of 10 inflammatory cytokines employing the Mesoscale Discovery Platform (MSD, Rockville, MD, USA) electrochemiluminescence technology, with subsequent validation via enzyme-linked immunosorbent assay (ELISA).

Results

Distinct elevation of IL-6, IL-8, IL-10, and TNF-α levels was evident in the CSF of the SICH subgroup relative to controls. Elevated Glasgow Coma Scale (GCS) scores among SICH patients were concomitantly linked with diminished CSF concentrations of IL-6, IL-8, IL-10, and TNF-α, indicating an inverse relationship. Notably, CSF inflammatory cytokine concentrations were consistently elevated in SICH patients manifesting with hydrocephalus compared to those without. Elevations in IL-6, IL-8, IL-10, and TNF-α in the CSF were significantly pronounced in the poor prognosis cohort (Glasgow Outcome Scale, GOS 1–3) relative to their counterparts with a favorable prognosis (GOS 4–5). The AUC values for the aforementioned cytokines in predicting SICH prognosis were 0.750, 0.728, 0.717, and 0.743, respectively.

Conclusions

The initial CSF levels of IL-6, IL-8, IL-10, and TNF-α upon admission offer significant insights into the severity of neural damage and serve as robust indicators for prognostication in SICH patients.

intracerebral hemorrhage

inflammation

cerebrospinal fluid

cytokines

Intracerebral hemorrhage (ICH) represents a profoundly severe and fatal subtype of stroke, accounting for an estimated 10–15% of all stroke instances, characterizing it as a quintessential clinical emergency [1]. Despite notable advancements in the clinical management of ICH, particularly in the realm of hematoma surgical interventions, the prognosis for ICH patients remains distressingly bleak. A staggering mortality rate, approaching 50%, is observed, largely attributed to the cumulative ramifications of primary and secondary brain injuries [2]. Primary brain injuries predominantly emanate from hematoma compression and acute hydrocephalus induced by cerebrovascular rupture and consequent hemorrhage. Concomitantly, secondary brain injuries are orchestrated by the pivotal role of the inflammatory response, mediated by the activation of microglia [3]. Specifically, erythrocyte detritus and associated degradation byproducts instigate the activation of microglial cells. This activation, in turn, catalyzes the discharge of inflammatory cytokines, eliciting a robust inflammatory cascade [4] which inflicts considerable detriment on neural tissues, manifesting as neurobehavioral aberrations, cerebral edema, and breaches in the blood-brain barrier integrity [5–7]. Subsequent to an ICH episode, the compromised blood-brain barrier facilitates the migration of neurogenic inflammatory cytokines into systemic circulation, instigating a domino effect of inflammatory responses, potentially culminating in systemic inflammatory response syndrome and even multi-organ dysfunction [2, 8]. Hence, heightened cytokine expressions may serve as harbingers of central nervous system (CNS) deterioration post-ICH.

Historically, the pathophysiological intricacies of ICH have been scrupulously examined in experimental animal frameworks, with neuroinflammation emerging as a research frontier of immense therapeutic promise [9, 10]. Indeed, the neuroinflammatory cascade, activated in the wake of ICH, is surmised to be an imperative physiological counter-response to neural trauma [6]. During such ICH incidents, neural infiltration by blood constituents triggers both innate and adaptive immunological activations, thereby congregating peripheral blood leukocytes in hematoma vicinities [11, 12]. This leads to the secretion of pivotal inflammatory mediators like tumor necrosis factor-a (TNF-α), interleukins (IL), and chemokines, all of which are instrumental in orchestrating acute inflammatory sequelae post-neural injury [4]. A plethora of prior clinical investigations have accentuated the augmented levels of these inflammatory markers in ICH patient serum, establishing their potent association with neurological deficits, cerebral edema, and unfavorable clinical outcomes [13–16]. Given that cerebrospinal fluid (CSF) is intimately aligned with neural tissues, surveillance of alterations in inflammatory cytokine expressions, particularly their efflux from neural tissues into CSF, provides an invaluable insight into neuroinflammatory fluxes, mirroring the evolving stages of neural tissue or neuronal injury [17–19].

Consequently, the focal objective of this research endeavor is to meticulously dissect the interplay between alterations in CSF-residing inflammatory cytokine expressions in ICH patients and ICH progression, alongside discerning their correlation with the severity of neural compromise and neurological prognostication. The ultimate vision is to harness these insights to augment diagnostic precision and therapeutic interventions for ICH.

2. 1 Research Objects

A total of 100 patients, diagnosed with new-onset ICH and admitted to the General Hospital between September 2019 and January 2023, were enlisted as the study cohort. Inclusion Criteria: (1) Patients presenting with primary cerebral hemorrhage admitted within 24 hours post symptom onset; (2) Patients that met the rigorous diagnostic benchmarks for SICH as ascertained by comprehensive clinical evaluations including medical anamnesis, neurologic examinations, and imaging diagnostics [20]. Exclusion Criteria: (1) Individuals manifesting acute hemorrhage due to ruptured vascular malformations or aneurysms, or cerebral hemorrhage consequential to neoplastic growths or trauma; (2) Patients diagnosed with malignant neoplasms; (3) Individuals with pronounced organ dysfunction, spanning cardiac, hepatic, and renal systems; (4) Patients with a documented cerebrovascular event within the preceding three months; (5) Conditions such as congenital vascular malformations, vasculitis, and autoimmune diseases; (6) Recent intake of oral anticoagulant or antiplatelet therapeutics; (7) Patients demonstrating contraindications or unwillingness for CSF extraction. A comparative control group was constituted with 50 patients, devoid of CNS-related ailments, who sought treatment during the same temporal bracket. Inclusion Criteria for Control Group: (1) Absence of CNS-associated pathologies (encompassing cerebrovascular conditions, CNS infections, demyelinating and degenerative disorders) corroborated via cranial CT/MRI; (2) Normalcy in routine CSF diagnostics and biochemistry; (3) No antecedent history of cardiovascular or cerebrovascular diseases. Pertinent demographic, clinical, and laboratory data were meticulously curated from each participant cluster.

2. 2 CSF Acquisition

CSF samples were procured via lumbar puncture and subsequently dispatched for diagnostic assays as mandated clinically post-admission. For the purposes of this study, CSF samples were harvested on the inaugural admission event, with standard and biochemical evaluations conducted in strict adherence to the prescribed Standard Operating Procedures (SOPs). Subsequent to collection, CSF aliquots underwent centrifugation at 3,000 RPM for a duration of 10 minutes at a controlled temperature of 4℃ and were then stored at a cryogenic temperature of -80℃.

2. 3 Inflammatory cytokine detection

For preliminary screenings, CSF specimens from 27 ICH patients and 15 control subjects were assayed to quantify the expression dynamics of an array of cytokines: IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12P70, IL-13, TNF-α, and interferon (INF)-γ utilizing the robust Mesoscale Discovery Platform (MSD, Rockville, MD, USA) electrochemiluminescence platform. The intrinsic merit of the MSD technology lies in its frugality in sample volume requisites coupled with its capacity for expeditious multiplexed analysis. The operational crux of MSD is underpinned by the formation of a bipartite antibody sandwich complex, encompassing a capture antibody and a detection antibody conjugated with ruthenium tricyclic, both binding the target analyte. Upon electrical stimulation, the ruthenium tricyclic undergoes a chemiluminescent reaction, emitting photons whose intensity linearly mirrors the target concentration. In light of the discernible amplification of inflammatory cytokines observed during the MSD preliminary screenings vis-à-vis controls, subsequent efforts concentrated on augmenting the sample size for validation. CSF specimens from 100 ICH and 50 control patients were meticulously assayed employing specific enzyme-linked immunosorbent assay (ELISA) kits: IL-6 (Ref: E-EL-H6156), IL-8 (Ref: E-EL-H6008), IL-10 (Ref: E-EL-H6154), and TNF-α (Ref: E-EL-H0109c) sourced from Wuhan Elite Bioscience and Technology Co, Ltd. The assays were executed in rigorous conformity with the manufacturer's guidelines. The absorbance at 450nm (OD450) was determined post colorimetric reactions, enabling the computation of target cytokine concentrations through the standard curve methodology.

2. 4 Judgement criteria of disease severity

The assessment of disease severity in ICH patients upon admission was executed in alignment with the Glasgow Coma Scale (GCS) [21]. Scores spanning 9-15 were designated as indicative of mild to moderate disease manifestation, while scores within the range of 3-8 were interpreted as severe disease presentations.

2. 5 Diagnosis of acute hydrocephalus

Cerebral CT scans were meticulously acquired for patients diagnosed with ICH. The establishment of hydrocephalus was contingent upon the observation of any of the following radiographic hallmarks: (1) overt dilation of the temporal horn, (2) pronounced bulging of the third ventricular wall, (3) the rounding-off of the frontal horn, (4) the obliteration of cerebral sulci, and (5) ventricular enlargement that is incongruous with sulcal dilation. The presence of any of these criteria within the cerebral CT scan affirmed the diagnosis of acute hydrocephalus.

2. 6 Prognostic judgement criteria

A longitudinal follow-up was conducted for ICH patients, culminating six months post-discharge. Prognostic outcomes were meticulously evaluated employing the Glasgow Outcome Scale (GOS) [22]. A GOS score situated between 4 and 5 (where 4 represents mild disability and 5 signifies good recovery) was construed as a favorable prognosis. Conversely, a GOS score between 1 and 3 (1 indicative of mortality, 2 delineating a vegetative state, and 3 suggesting severe disability) was demarcated as a prognosis of concern.

2. 7 Data statistics

For data analytics, the study deployed software platforms, namely SPSS (Version 26. 0) and GraphPad Prism (Version 8. 0. 2). Categorical variables were represented as case counts, and intergroup comparisons were made employing the χ2 test. Quantitative datasets adhering to the normal distribution paradigm were articulated as mean ± standard deviation, with the t-test employed to discern differences across continuous variables spanning two distinct cohorts. Data sets not conforming to normal distribution were depicted as median with interquartile range (M [P25, P75]), and comparisons between the two groups were made utilizing the Mann-Whitney U test. Associations were probed through Spearman's rank correlation coefficient analysis. Receiver Operating Characteristic (ROC) curves were sketched to decipher the prognostic predictive value of cytokine levels, specifically IL-6, IL-8, IL-10, and TNF-a, in the ICH cohort. A p-value below 0.05 was deemed indicative of statistical significance.

3. 1 Clinical data of study subjects

Based on the stipulated inclusion criteria, 100 ICH-afflicted patients and 50 controls were meticulously selected. Within the ICH cohort, 65 individuals were male and 35 were female, boasting an average age of 58.52 ± 13.22 years. The median GCS score for this group stood at 9 (IQR, 5-12). Conversely, the control group encompassed 36 males and 14 females, having an average age of 31.02 ± 15.00 years. A comprehensive juxtaposition of demographic and clinical indices is encapsulated in Table 1.

Table 1 Demographic and Baseline Characteristics.

Characteristic	Control group (n=50)	ICH group (n=100)	P value
sex, n (%)			0.389
Female	14(28)	35(35)
male	36(72)	65(65)
Age, yr, mean (SD)	31.02(15.00)	58.52(13.22)	<0.0001*
BMI(Kg/m2), median (IQR)	24.22(23.39-26.23)	24.22(23.43-26.03)	0.484
Hypertension, n (%)	12(24)	55(55)	<0.001*
Diabetes, n (%)	4(8)	12(12)	0.454
Hematoma location, n (%)			N/A
Basal ganglia	N/A	45(45)
Lobar	N/A	14(14)
Thalamus	N/A	26(26)
Cerebellum	N/A	6(6)
Brainstem	N/A	3(3)
Ventricle	N/A	6(6)
Into the ventricle, n (%)	N/A	45(45)	N/A
Final ICH volume(ml), n (%)			N/A
<30	N/A	52(52)
≥30	N/A	48(48)
Hydrocephalus, n (%)	N/A	16(16)	N/A
GCS, median (IQR)	N/A	9(5-12)	N/A
Time from symptom onset to MIS (hours), mean (SD)	N/A	4.15(3.62)	N/A
Infection while in hospital, n (%)	N/A	27(27)	N/A
Routine examination of CSF
WBC(n), median (IQR)	3(2-6)	20(6-233)	<0.0001*
RBC(n), median (IQR)	6(3.75-10)	3580(212.5-13100)	<0.0001*
Biochemical reactions of CSF
PRO(g/L), median (IQR)	0.245(0.190-0.310)	1.10(0.53-2.20)	<0.0001*
GLU (mmol/L), median (IQR)	3.315(3.133-3.603)	3.830(3.180-5.640)	<0.001*
CL (mmol/L), median (IQR)	121.4(120.4-122.8)	122.5(117.6-127.3)	0.471
GOS, n (%)			N/A
1~3	N/A	53(53)
4~5	N/A	47(47)

Abbreviations: GCS, Glasgow coma scale; ICH, Intracerebral hemorrhage; IQR, Interquartile range; MIS, minimally invasive surgery; SD, standard deviation; BMI, Body Mass Index; CSF, Cerebrospinal fluid; WBC, White blood cell; RBC, Red blood cell; PRO, protein; GLU, glucose; GOS, Glasgow outcome scale. * Statistically significant.

3. 2 Elevated levels of inflammatory cytokine expression in CSF of ICH patients

The CSF of ICH patients was assayed for inflammatory cytokine content utilizing the MSD electrochemiluminescence technique. Herein, it was discerned that the concentrations of IL-6, IL-8, IL-10, and TNF-a were substantially elevated vis-à-vis the control cohort (P<0.05) (Fig. 1A-D). Cytokines not exhibiting statistically pertinent differentials have been omitted from representation. Given the relatively petite sample size of the preliminary screening, potential biases might taint the results, underscoring the imperative to validate findings via an augmented sample size.

Fig. 1

In light of the aforementioned revelations, the cohort sizes of ICH patients and controls were amplified for a more granular assessment of inflammatory cytokine expression in the CSF via ELISA. In juxtaposition with controls, the ICH patients demonstrated significantly elevated levels of IL-6, IL-8, IL-10, and TNF-a in their CSF, each bearing statistical significance (P<0.05) (Fig. 2A-D).

Fig. 2

3. 3 Disease severity of ICH correlates with inflammatory cytokine levels in the CSF

Elevated expressions of IL-6, IL-8, IL-10, and TNF-a were discerned in the CSF of patients with GCS scores bracketed between 3-8, vis-à-vis their counterparts with GCS scores spanning 9-15 (P<0.05) (Fig. 3A-D). Delving deeper into the intricate relationship between ICH severity and inflammatory cytokine levels within the CSF, a comprehensive correlation analysis was embarked upon. The outcomes of this investigation illuminated that CSF levels of IL-6, IL-8, IL-10, and TNF-a were all inversely associated with GCS scores (r=-0.38, p<0.001; r=-0.34, p<0.001; r=-0.28, p<0.01; r=-0.24, p<0.05). This intimates that as the clinical severity—denoted by an escalating GCS—mitigates, there's a concomitant decrement in the expression of these inflammatory cytokines within the CSF (Fig. 4A-D).

Fig. 3

3. 4 Higher levels of inflammatory cytokines in CSF of patients with hydrocephalus

The quantified levels of IL-6, IL-8, IL-10, and TNF-a within the CSF of patients diagnosed with both ICH and hydrocephalus surpassed the concentrations in patients devoid of hydrocephalus (P<0.0001, P<0.05, P<0.001, and P<0.001 respectively) as illustrated in Fig. 5A-D.

Fig. 4

3. 5 Relationship between inflammatory cytokine expression levels in CSF of ICH patients and prognosis of ICH

Patients were meticulously tracked over a 6-month span and subsequently grouped based on their prognosis: the favorable prognosis set (GOS between 4 and 5) and the unfavorable prognosis collection (GOS ranging from 1 to 3). Initial univariate scrutiny indicated that several factors—namely age, hemorrhagic volume, coexisting ailments, GCS, total protein content, and glucose concentration within the CSF—significantly influenced ICH prognostic outcomes (refer to Table 2). CSF concentrations of IL-6, IL-8, and TNF-a in the cohort exhibiting poor prognostic outcomes surpassed their counterparts in the favorable prognosis category. Conversely, CSF levels of IL-10 were subdued in the favorable prognosis cluster when compared to the unfavorable prognosis grouping (depicted in Fig. 6A-D). ROC examination highlighted that the area under the receiver operating characteristic curve (AUC) for inflammatory markers IL-6, IL-8, IL-10, TNF-a, and their collective predictive potency for prognosis within ICH patients' CSF stood at 0.750, 0.728, 0.728, 0.717, 0.743, and 0.874 respectively. This was in conjunction with sensitivities of 92.5%, 52.8%, 67.9%, 47.2%, and 84.9%, and specificity rates of 48.9%, 80.9%, 70.2%, 91.5%, and 76.6%, as visualized in Fig. 7 and elucidated in Table 3.

Fig. 5

Table 2 Univariate analysis for the clinical characteristics of subjects with a favorable or unfavorable outcome

Characteristic	Favorable (n=47)	Unfavorable (n=53)	P value
sex, n (%)			0.515
Female	18(38.3)	17(32.1)
male	29(61.7)	36(67.9)
Age, yr, mean (SD)	55.28(13.67)	61.40(12.22)	<0.05*
BMI(Kg/m2), median (IQR)	24.22(22.49-26.45)	24.00(23.43-26.10)	0.966
Hypertension, n (%)	21(44.7)	34(64.2)	<0.05*
Diabetes, n (%)	1(2.1)	11(20.8)	<0.01*
Hematoma location, n (%)			0.248
Basal ganglia	17(36.2)	28(52.8)
Lobar	9(19.1)	5(9.4)
Thalamus	12(25.5)	14(26.4)
Cerebellum	5(10.6)	1(1.8)
Brainstem	1(2.1)	2(3.8)
Ventricle	3(6.4)	3(5.7)
Into the ventricle, n (%)	17(36.2)	28(52.8)	0.095
Final ICH volume(ml), n (%)			<0.001*
<30	34(72.3)	18(34)
≥30	13(27.7)	35(66)
Hydrocephalus, n (%)	6(12.8)	10(18.9)	0.406
GCS, median (IQR)	11(9-14)	6(5-8)	<0.0001*
Time from symptom onset to MIS (hours), mean (SD)	4.091(4.302)	4.235(2.547)	0.176
Infection while in hospital, n (%)	10(21.3)	17(32.1)
Routine examination of CSF
WBC(n), median (IQR)	3040(160-9190)	4280(225-18540)	0.213
RBC(n), median (IQR)	8(4-120)	80(6-420)	0.073
Biochemical reactions of CSF
PRO(g/L), median (IQR)	0.78(0.44-1.44)	1.64(0.6-2.645)	<0.01*
GLU (mmol/L), median (IQR)	3.47(3.080-4.88)	4.605(3.3-6.323)	<0.05*
CL (mmol/L), median (IQR)	121.8(117.1-125.6)	122.9(118.1-131.5)	0.134

Abbreviations: GCS, Glasgow coma scale; ICH, Intracerebral hemorrhage; IQR, Interquartile range; MIS, minimally invasive surgery; SD, standard deviation；BMI，Body Mass Index; CSF, Cerebrospinal fluid; WBC, White blood cell; RBC, Red blood cell; PRO, protein; GLU, glucose. * Statistically significant.

Table 3 The expression levels of IL-6, IL-8, IL-10 and TNF-a in CSF and the efficacy indexes of the four factors in predicting ICH prognosis

Cytokines	AUC	Sensitivity (%)	Specificity (%)	95%CI	Youden index	Cutoff value (pg/ml)	p
IL-6	0.750	92.5	48.9	0.654-0.845	0.414	198.01	<0.0001
IL-8	0.728	52.8	80.9	0.632-0.825	0.337	1756.64	<0.0001
IL-10	0.717	67.9	70.2	0.616-0.818	0.318	1.58	<0.001
TNF-α	0.743	47.2	91.5	0.648-0.839	0.387	10.72	<0.0001
Combine	0.874	84.9	76.6	0.806-0.941	0.615	—	<0.0001

In the current medical landscape, ICH management remains a formidable challenge, and the associated mortality and morbidity rates are distressingly high [23]. The imperative of accurately gauging neurological impairment and prognostication in ICH patients during early stages cannot be overstated. Facilitating prompt and effective interventions is pivotal in curtailing morbidity, mortality, and disability rates for these patients. Notably, ICH precipitates a cascade of intricate pathophysiological reactions in the body. The presence of blood within cerebral confines engenders an inflammatory onslaught, constituting a crucial underpinning for both immediate and protracted cerebral damage post-ICH. This mechanism also hints at a prospective therapeutic target [19, 24]. Previous animal-based experiments and clinical endeavors have underscored the salience of post-ICH inflammatory reactions in influencing the restoration of neurological functionality and the overall prognosis of ICH. Inflammatory cytokines stand as critical intermediaries in the neuroinflammatory aftermath of ICH, with cytokines such as IL-6, IL-10, and TNF-α playing pivotal roles in orchestrating immune and central nervous system activities [13, 25–27].

Our investigation reveals a marked elevation in the CSF levels of inflammatory cytokines IL-6, IL-8, IL-10, and TNF-α upon initial admission of ICH patients, whereas cytokines such as IL-1β, IL-2, IL-4, IL-12P70, IL-13, and INF-γ did not manifest discernible disparities. Contemporary literature posits that aseptic inflammatory reactions emerge as typical manifestations in patients grappling with ventricular hemorrhage in the days succeeding the hemorrhagic event. A pronounced inflammatory reaction is directly proportional to the initial hemorrhage magnitude, corroborated by an augmented leukocyte count in CSF adjusted for hemorrhage [17, 28]. This observation harmonizes with the inflammatory cytokine trends discerned in our study. Case analyses pertaining to aneurysmal subarachnoid hemorrhage have similarly reported augmented CSF inflammatory cytokine levels, encompassing IL-1α, IL-1β, IL-2, IL-4, IL-6, INF-γ, and TNF-α [29–31]. Huang et al. [32] elucidated that elevated CSF S100B levels in ICH patients might potentiate IL-1β expression in primary microglia via pathways like ERK1/2, JNK, and p38 MAPK. This pro-inflammatory cytokine, IL-1β, triggers an acute-phase reaction in the CNS, indicating a potential role of the cerebral injury biomarker S100B in modulating inflammatory dynamics following cerebral hemorrhage. Concurrently, Wendy et al. [33] documented post-ventricular hemorrhage amplification of CSF IL-1β, IL-6, IL-10, TNF-α, and CCL2 expressions, which then manifested a decline in subsequent evaluations. Analogously, elevated IL-8 levels were discerned in the CSF of neonates subjected to ventricular hemorrhage [34, 35]. Animal model investigations of ventricular hemorrhage have echoed these observations [36–38]. Collectively, these studies robustly affirm the manifestation of a neuroinflammatory cascade in cerebral tissues post-ICH. Nonetheless, the precise cytokine portfolio evidenced in the CSF reflecting this inflammatory dynamic isn't in total alignment with our study. Potential underpinnings for such variations could be attributed to differences in hemorrhage location, volume, and the temporal evolution of the disease among the sampled subjects, necessitating more granular research.

Following an ICH, there exists a pronounced correlation between inflammatory cytokine concentrations in the CSF and the extent of cerebral tissue damage. Wendy et al. [33] delineated a significant association between the CSF concentrations of IL-6, IL-8, and IL-10, and the volume of cerebral hemorrhage. Additionally, a discernible linkage was observed between the admission GCS score and the concentrations of TNF-α and IL-8. In a similar vein, Yan et al. [39] elucidated that the levels of TNF-α, IL-6, and IL-1β in the blood or CSF of individuals with acute spontaneous ICH were substantially elevated when juxtaposed with those of patients with mild ICH or healthy counterparts. Conversely, Belarbi et al. [40] indicated that augmented levels of IL-11, TNF-α, and vascular endothelial growth factor were congruent with elevated neurologic deficit scores, suggestive of profound neurologic damage. Our present analysis discerned that in ICH patients, those with a diminished GCS showcased elevated CSF concentrations of IL-6, IL-8, IL-10, and TNF-α. Subsequent correlation evaluations further affirmed that the expression levels of IL-6, IL-8, IL-10, and TNF-α were inversely proportional to the GCS. This observation resonates, albeit not entirely, with the findings detailed in extant literature. Given that the CSF constitutes an extracellular fluid of the CNS, its analysis provides invaluable insights into the CNS's state. Such revelations underscore the potential of leveraging CSF inflammatory cytokine levels as an index of cerebral damage post-ICH, facilitating an informed assessment of the ICH patient's status.

Hydrocephalus emerges as a sequela of intraventricular and subarachnoid hemorrhages. Katherine et al. [41] embarked on elucidating the neuroinflammatory dynamics underpinning hydrocephalus post-intraventricular hemorrhage, concluding that inflammatory cytokines are central players in this pathway. Gakwaya et al. [42], in their exploration, ascertained that cytokine concentrations, specifically TNF-α, IL-1α, IL-4, IL-6, and IL-12, were markedly escalated in the CSF of neonates presenting with posthemorrhagic hydrocephalus. It's pivotal to underscore that the CNS of preterm neonates remains in a nascent developmental phase, suggesting potential disparities when compared to adults. In our investigation, we endeavored to delineate the relationship between hydrocephalus and CSF inflammatory cytokine concentrations in ICH patients. Our findings highlight that ICH patients with hydrocephalus exhibited elevated CSF levels of IL-6, IL-8, IL-10, and TNF-α compared to their counterparts devoid of hydrocephalus. This intimates that cerebral hemorrhages, when concomitant with hydrocephalus, intensify the neuroinflammatory cascade, exacerbating cerebral tissue damage and consequently influencing patient prognosis.

The nexus between neuroinflammatory response and prognosis subsequent to ICH remains somewhat ambiguous. While a pair of investigations focused on spontaneous intraventricular hemorrhage found no discernible correlation between the aseptic inflammatory response in the CSF and unfavorable prognosis [17, 18], another inquiry [39] highlighted that ICH patients exhibiting elevated concentrations of TNF-α in the blood or CSF had a 1.06-fold increased likelihood of an adverse outcome, compared to their counterparts with reduced TNF-α levels. Furthermore, patients with augmented IL-6 concentrations had a predilection towards unfavorable outcomes, amplifying their odds by a factor of 61.6 compared to those with diminished IL-6 concentrations. This underscores the utility of measuring TNF-α and IL-6 concentrations in prognostic evaluations for ICH patients. In investigations centered on subarachnoid hemorrhage, it has been evinced that CSF inflammatory cytokines might serve as potent prognostic biomarkers [43–45]. Serum concentrations of inflammatory cytokines such as IL-4, IL-6, and IL-10 in ICH patients exhibit significant correlations with functional outcomes, and modulation of these cytokines ostensibly ameliorates ICH prognosis [13, 25, 26, 46]. In our current analysis, we delved into the association between CSF inflammatory cytokine levels and prognosis in ICH patients, revealing that, six months post-hospital discharge, admission levels of IL-6, IL-8, IL-10, and TNF-α were notably elevated in the poor-prognosis cohort compared to the favorable-prognosis cohort. This intimates that these cytokine levels in the CSF not only influence ICH prognosis but that heightened admission levels of CSF IL-6, IL-8, IL-10, and TNF-α are emblematic of an elevated risk of adverse prognosis. Furthermore, this buttresses the hypothesis that tempering the neuroinflammatory cascade post-ICH and curtailing inflammatory cytokine expression could potentially attenuate cerebral tissue damage [47–50] and enhance patient prognosis. Concurrently, our ROC curve analysis posits that CSF levels of IL-6, IL-8, IL-10, and TNF-α might be superior prognostic indices for ICH.

This study is not without limitations. Foremost, our ICH cohort predominantly consisted of elderly participants, whereas the control group was largely characterized by younger individuals, which, given the paucity of suitable cases, could introduce an age-related bias. Additionally, given the variability in CSF collection timings and intervals for ICH patients, our analysis was circumscribed to the initial CSF samples obtained upon hospital admission, in relation to disease severity and prognosis. Lastly, we did not incorporate analyses of inflammatory cytokines from concurrent blood samples. Given these lacunae, future iterations of this study aim to augment the clinical case count and undertake an expansive multicenter investigation for enhanced validation.

Our findings elucidate that the CSF expression levels of IL-6, IL-8, IL-10, and TNF-α in ICH patients inversely correlate with patient GCS. Moreover, the admission levels of these cytokines in the CSF were significantly augmented in the cohort with a suboptimal prognosis at the six-month post-discharge follow-up compared to their favorable-prognosis counterparts. This suggests that CSF inflammatory cytokine expression levels could be pivotal in gauging the extent of cerebral injury and prognosticating outcomes in ICH patients, thereby offering a robust reference for ICH clinical diagnosis and therapeutic interventions.

Acknowledgements

Not applicable.

Authors’ contributions

QS and ZZ were corresponding author and they were responsible for experimental design. TG and JP performed ELISA experiments. LC and KL performed all calculation and data analysis. LW was responsible for samples collection. All authors read and contributed to the manuscript.

Data Availability

All data reported in this manuscript will be made available from the corresponding author (Qinghai Shi (email: [email protected])) on reasonable request.

Funding

This work was supported by the National Natural Science Foundation of China [grant number 81871020].

Declaration

Competing Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Ethics approval and consent to participate

The protocol, which adhered to the principles of the Declaration of Helsinki, received approval from the Ethics Committee of the General Hospital of Xinjiang Military Command, under the reference number 2023RR0301. All patients provided their written informed consent.

Consent for publication

Not applicable.

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No competing interests reported.

Association of inflammatory cytokines expression in cerebrospinal fluid with the severity and prognosis of spontaneous intracerebral hemorrhage

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