Leukotriene-mediated neuroinflammation, toxic brain damage, and neurodegeneration in acute methanol poisoning

Abstract

Context: The role of neuroinflammation in methanol-induced toxic brain damage has not been studied.

Objective: We studied acute concentrations and the dynamics of leukotrienes (LT) in serum in hospitalized patients with acute methanol poisoning and in survivors.

Methods: Series of acute cysteinyl-LT and LTB4 concentration measurements were performed in 28/101 hospitalized patients (mean observation time: 88 ± 20 h). In 36 survivors, control LT measurements were performed 2 years after discharge.

Results: The acute maximum (C_max) LT concentrations were higher than concentrations in survivors: C_max for LTC4 was 80.7 ± 5.6 versus 47.9 ± 4.5 pg/mL; for LTD4, 51.0 ± 6.6 versus 23.1 ± 2.1 pg/mL; for LTE4, 64.2 ± 6.0 versus 26.2 ± 3.9 pg/mL; for LTB4, 59.8 ± 6.2 versus 27.2 ± 1.4 pg/mL (all p < 0.001). The patients who survived had higher LT concentrations than those who died (all p < 0.01). Among survivors, patients with CNS sequelae had lower LTE4 and LTB4 than did those without sequelae (both p < 0.05). The LT concentrations increased at a rate of 0.4–0.5 pg/mL/h and peaked 4–5 days after admission. The patients with better outcomes had higher cys-LTs (all p < 0.01) and LTB4 (p < 0.05). More severely poisoned patients had lower acute LT concentrations than those with minor acidemia.

The follow-up LT concentrations in survivors with and without CNS sequelae did not differ (all p > 0.05). The mean decrease in LT concentration was 30.9 ± 9.0 pg/mL for LTC4, 26.3 ± 8.6 pg/mL for LTD4, 37.3 ± 6.4 pg/mL for LTE4, and 32.0 ± 8.8 pg/mL for LTB4.

Conclusions: Our findings suggest that leukotriene-mediated neuroinflammation may play an important role in the mechanisms of toxic brain damage in acute methanol poisoning in humans. Acute elevation of LT concentrations was moderate, transitory, and was not followed by chronic neuroinflammation in survivors.

Keywords:

• Neuroinflammation
• leukotrienes
• methanol poisoning
• brain damage
• sequelae of poisoning
• nontraumatic brain injury

Introduction

Background

Methanol is one of the most widely used toxic alcohols throughout the world. Mass or cluster acute methanol poisonings as a result of its use as a cheap substitute for ethanol occur frequently globally, mainly in developing countries [1]. Recent mass poisoning outbreaks in Estonia with more than 150 cases [2], in Norway with more than 50 cases [3], and in the Czech Republic with more than 120 cases [4] provide clear evidence of this public health emergency for the health systems of developed European countries as well. If therapeutic interventions are inadequate or delayed, mortality exceeding 40% as well as serious long-term visual and CNS sequelae in survivors may occur [5,6].

Importance

Bilateral necrosis of the basal ganglia and necrotic lesions in the subcortical white matter are typical magnetic resonance (MRI) signs of brain damage in acute methanol poisoning [7–9]. Methanol-induced optic neuropathy may resolve within a few weeks with complete recovery [10]. However, long-term visual sequelae may persist in up to 25–40% of patients [11–13].

The mechanisms of acute brain and optic nerve damage in acute methanol poisoning are not well understood. In humans, methanol is oxidized by alcohol dehydrogenase (ADH) to formaldehyde and then by aldehyde dehydrogenase to formic acid [14]. As a moderate inhibitor of mitochondrial cytochrome c oxidase (Ki ∼6 mmol/L), formic acid impairs tissue utilization of oxygen resulting in excess lactic acid production and depletion of ATP in cells [15,16]. Nevertheless, there is no difference in the serum formic acid concentrations in patients with lethal outcome and in survivors with sequelae of poisoning [17]. MRI signs of brain damage can be found in survivors of poisoning with low serum formic acid concentration on admission (2–4 mmol/L), while the patients with high serum formic acid concentration on admission of about 15 mmol/L may survive without CNS sequelae [9].

Neuroinflammation is one of the neuroprotective mechanisms associated with repair and recovery of the brain after acute traumatic and non-traumatic brain injury such as stroke, hypoxia, ischemia, and poisoning [18]. Leukotrienes (LTs) are the main mediators of neuroinflammation [19,20]. In the brain, LTB4 and cysteinyl leukotrienes (cys-LTs: LTC4, LTD4, and LTE4) are produced in response to a variety of acute brain injuries and have been related to brain edema development and to disruption of the brain–blood barrier (BBB) [21,22]. LTs exert their biological activities through G protein-coupled receptors, cys-LT1 and cys-LT2 for cys-LTs and BLT 1 and BLT 2 for LTB4. The cys-LT1 receptor mediates astrocyte proliferation after brain ischemia, while the cys-LT2 receptor plays an important role in regulating the response on cytotoxic effects of ischemic injury [22,23].

In experimental animal studies, it was demonstrated that closed head injury had resulted in a marked increase in activated microglia and reactive astrocytes persisting for as long as 1 month [24,25]. Activated microglia produced LTs, and later cytokines and chemokines, which recruit other immune cells that enhance the neuroinflammatory response [25–27]. These mediators of neuroinflammation reach the peripheral circulation either through BBB disruption or via the glymphatic system in the brain with an intact BBB [28].

The role of endogenous neuroinflammatory mechanisms in the pathophysiology of non-traumatic methanol-induced brain damage has not been studied. Brain lesions in patients with severe methanol poisoning pass through several stages during one to two weeks: general or multifocal subcortical white matter and pericapsular or focal basal ganglia edema followed by foci of non-hemorrhagic necrosis, which can progress to vast hemorrhagic necrotic lesions [9]. Formic acid in the brain possibly acts as a trigger initiating a complex array of pathologic and protective mechanisms including neuroinflammatory reaction mediated by LTs and the interaction of these mechanisms may determine the outcome of poisoning and the degree of brain and optic nerve damage in survivors. At present, LTs in peripheral blood serum as markers of acute neuroinflammation in methanol poisoning were not measured in humans.

Aim of the study

In this study, we examined the acute concentrations and dynamics of LT concentration changes in peripheral blood serum in patients with confirmed methanol poisoning during hospital treatment and tested the ability of these markers to classify favorable versus unfavorable (death or long-term visual/CNS sequelae) outcome of poisoning. Further, we measured LT concentrations in survivors 2 years after discharge from hospital to test their association with long-term health sequelae of methanol poisoning. We hypothesized that acute neuroinflammation would result in the elevation of LT concentrations in peripheral blood serum that would classify subject outcomes at clinically relevant time points.

Materials and methods

Study design and setting

This was a longitudinal cohort study of patients with confirmed acute methanol poisoning treated in hospitals during the Czech Republic mass methanol poisoning outbreak in 2012 [4]. The admission clinical and laboratory data were collected prospectively by the treating providers using a standardized data collection form and sent to the Toxicological Information Center (TIC) on the day following each admission to hospital. The data on hospital treatment and outcome were collected and reviewed from hospital discharge reports. The follow-up clinical examination of survivors was performed two times during the study period: 3–8 months and 2 years after discharge from the hospital.

The study was performed in 30 hospitals in 11 regions of the Czech Republic, where the poisoned patients had been treated. The follow-up clinical examination of survivors was performed in the General University Hospital in Prague. The measurements of blood serum LT concentration were performed in the Laboratory of Medicinal Diagnostics, University of Chemistry and Technology in Prague.

The study was approved by the General University Hospital Ethics Committee in Prague, Czech Republic.

Selection of participants and treatment

To identify the cases for the study, mandatory reporting to the Ministry of Health and the Czech Republic TIC on all cases of hospital admission with laboratory confirmed methanol poisoning and nationwide daily monitoring of the situation in all hospitals started on 6 September 2012, 3 days after admission of the first three patients with acute methanol poisoning. All patients hospitalized with confirmed acute methanol poisoning were eligible for this study. Excluded were patients who died out of hospital before admission and hospitalized patients, if blood samples for serum LTs measurement were not taken on admission and during hospital treatment. Further, the patients with less than four blood samples collected during hospital treatment were excluded from the study.

The patients were treated in accordance to the American Academy of Clinical Toxicology (AACT) and European Association of Poisons Centres and Clinical Toxicologists (EAPCCT) practice guidelines for the treatment of methanol poisoning [29]. Bicarbonate was given as a buffer to the patients with severe acidemia on admission. Ethanol or fomepizole was used as antidotes to block ADH enzyme [30–32]. Intermittent hemodialysis (IHD) or continuous veno-venous hemodialysis/hemodiafiltration (CRRT) was performed to eliminate formic acid and methanol and to correct academia [33,34]. Folates were administered to substitute the internal pool (folinic or folic acid) [35]. No corticosteroids and nonsteroid anti-inflammatory drugs were administered.

Clinical examinations and laboratory analyses during hospital treatment

Detailed histories of poisoning and of ocular and systemic toxicity were obtained directly from the patients or from relatives of critically ill patients upon admission to hospital. Diagnosis was established when (i) a history of recent ingestion of illicit spirits was available and serum methanol was higher than 200 mg/L, or (ii) there was a history or clinical suspicion of methanol poisoning, and serum methanol was above the limit of detection with at least two of the following: pH <7.3, serum bicarbonate <20 mmol/L, or anion gap (AG) ≥20 mmol/L [29].

The clinical examination protocol included complete ocular examination with standard ophthalmologic tests (visual acuity, color vision, visual fields, contrast sensitivity, and fundus examination), cerebral computed tomography (CT) or MRI of the brain, and standard neurological examination (physical examination, motor, sensory, cerebellar, cranial nerves, and reflexes). Patients were considered to have visual sequelae of acute methanol poisoning if the symptoms of toxic neuropathy of the optic nerve were documented on admission and/or during hospitalization, with pathologic findings on visual acuity, perimeter, color vision, contrast sensitivity, and persisting lesions on fundoscopy on discharge. Similarly, patients were considered as having CNS sequelae of poisoning if symmetrical necrosis and hemorrhages of basal ganglia and subcortical white matter compatible with the diagnosis of acute methanol poisoning were present on CT or MRI of the brain.

Laboratory measurements of LTs concentration in peripheral blood serum

Venous blood samples for LTs measurements in peripheral blood serum were obtained on admission, each 2–4 h during the first 48 h of hospitalization, and each 6–12 h during further days of hospitalization. On the follow-up clinical examination of controls, venous blood samples were obtained prior to the clinical examination program. Blood samples were spun, serum separated, and frozen at −80° C until analyses.

Analyses of LTs (LTB4, LTC4, LTD4, and LTE4) were performed as previously described [36]. The method consists of a pretreatment step, solid-phase extraction for rapid and effective isolation of biomarkers from blood serum, and a detection method using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

Follow-up clinical examination protocol

The follow-up clinical examination protocol in survivors 3–8 months and 2 years after discharge included complete ocular examination and standard ophthalmic tests, optical coherence tomography (OCT) with retinal nerve fibers layer (RNFL) thickness evaluation, visual evoked potentials (VEP), MRI of the brain, neurological and neuropsychological examinations, including the Mini-Mental State Examination (MMSE), Natural history and neuroprotection in Parkinson plus syndromes – Parkinson plus scale (NNIPPS-PPS), motor, sensory, cerebellar, cranial nerves, and reflexes, biochemical tests (electrolytes, glucose, glycohemoglobin, albumin, pre-albumin, renal and hepatic tests, cholesterol, lipids, thyroid-stimulating hormone (TSH), vitamins B₁₂ and B₁, carbohydrate-deficient transferrin (CDT), ethyl glucuronide in urine, and standardized questionnaire forms. The examiners were masked to the serum LTs, methanol, and formic acid concentration, severity of poisoning, clinical course, treatment measures and clinical outcomes on discharge from hospital, as well as to the results of follow-up examinations.

RNFL thickness was measured on the OCT Spectralis Tracking Laser Tomography (Heidelberg Engineering GmbH, Heidelberg, Germany, software version 5.8.3) and compared to the normative database. On the diagram, the green area represented the mean RNFL thickness of normal eyes. Normal eyes were defined as those that fell within 5–95 percentile of normal distribution. Measured values in this range were considered “within normal limits”. The red area represented values below the first percentile of RNFL thickness of normal distribution. Measured values at this range were considered “abnormal – outside normal limits”. The yellow area represented values between the 1–5 percentile of normal distribution. Measured values in this range were considered “borderline”, and did not indicate that measured values related to disease state. The results of RNFL measurement 3–8 months and 2 years after discharge were compared to detect significant loss of RNFL thickness during the follow-up period. The criterion for the significant abnormal loss of RNFL was the loss of at least 2–4 mcm in all segments and/or loss of at least 4–6 mcm in one (mostly temporal) segment of retina detected on the second examination.

The VEP examination was performed on a two-channel TruTrace 4 Alien Technik CZ device. Monocular checkerboard pattern-reversal stimulation was used, with frequency of 1.5 c/s, angular size of the monitor 6° × 5° from the fixation point, angular size of checkerboard squares 40′. Luminance of the white and black squares was 84 cd/m² and 57 cd/m², respectively. Bandwidth of the amplifier was 1 Hz–1 kHz, the evoked response was registered from the Oz-Fz derivation. At each eye, the examination was performed twice in order to check reproducibility of the evoked complex. We evaluated latencies of waves N1, P1, and N2, and amplitudes N1P1 and P1N2. The measured values in patients were compared with our laboratory reference values determined as the central 95% interquartile interval of data measured on a group of 30 healthy individuals. Four criteria of abnormality were chosen: (1) missing evoked response, (2) wave P1 latency >117 ms, (3) interocular difference of waves P1 latencies >6 ms, and (4) amplitude of evoked complex <3 μV. The result was categorized as abnormal if at least one of the above-mentioned four criteria was fulfilled.

The best-corrected visual acuity (BCVA) was established on standard Snellen charts at 6 m (20 ft) distance. The visual acuity was considered pathologic if worse than 6/6. The visual field was examined by means of static perimetry (Medmont perimeter M700 automated perimeter (Medmont International Pty Ltd, Vermont, Australia, Neurological test, threshold strategy). The findings were considered pathologic if there were any defects in the visual field. The color vision was examined by means of Lanthony’s 15-D test (Richmond Products, Albuquerque, NM). The finding was considered normal up to three crossings, borderline with 3–7 crossings and pathologic with more than 7 crossings. The contrast sensitivity was examined by means of Pelli–Robson contrast sensitivity test (Clement Clarke International Ltd, Essex, UK). The finding was considered normal (1.35 and better), borderline (1.20–1.05) or pathologic (worse than 1.05). The fundus (posterior pole of the eye) was examined by means of biomicroscopy on the slit lamp with the +78 diopters lens (Volk-lens type). The finding was considered pathologic, if any related pathology of the optic nerve head and/or the adjacent retina was present).

All patients underwent brain MRI on Gyroscan Phillips 1.5 T system with the following protocol: axial T2-weighted image with slice thickness (THK) 6.0/0.6 mm through the whole brain, with parameters: repetition time (TR) 4241 ms, time to echo (TE) 100 ms, flip angle (FA) 90°, FLAIR (fluid attenuated inversion recovery): TR 11,000 ms, TE 140 ms, inversion time (TI) 2800 ms, FA 90°, T1-weighted image: TR 569 ms, TE 15 ms, FA 69°, T2-weighted image-fast field echo: TR 665 ms, TE 23 ms, FA 18°, single-shot diffusion-weighted image: TR 2901 ms, TE 75 ms, FA 90°, T1 weighted after administration of gadolinium and in coronal images centered to the orbital region T2-weighted image with suppression of fat (SPIR): TR 5506 ms, TE 100 ms, FA 90°. The patients were considered as having CNS sequelae of poisoning if symmetrical necrosis, with or without hemorrhages, of the basal ganglia (putamen, globus pallidus) and other brain lesions (brainstem, caudate nucleus, cerebellum, deposits in subcortical white matter, and optic nerve atrophy) compatible with the diagnosis of acute methanol poisoning were present on MRI scan of the brain.

Outcome of the study

The primary outcome of the study was acute LT concentration measured in peripheral blood serum in patients with methanol poisoning during hospital treatment, and the follow-up LT concentration measured in survivors 2 years after discharge from hospital. The secondary outcome was the association of serum LT concentration with laboratory parameters of severity of poisoning measured during the treatment, treatment modalities, and the outcome of poisoning, as well as with laboratory and clinical parameters studied on the follow-up examination, including the functions of optic nerve (results of VEP measurements), morphological changes of retina (results of RNFL measurements), and MRI signs of brain damage in survivors of poisoning.

Calculations and data analysis

Basic descriptive statistics (mean, median, CI, SD, skewness, and kurtosis) were computed for all variables, which were subsequently tested for normality using the Kolmogorov–Smirnov test. A χ² test was used to compare frequency counts of demographic and clinical categorical variables in the group of hospitalized patients with acute methanol poisoning and in the follow-up group. One-way analysis of variance with least significant difference was used to compare the concentration of LTs in peripheral blood serum. The bivariate relationship was assessed using a Pearson’s correlation coefficient. Statistical significance was set at p < 0.05. Statistical documentation was performed in Excel (Microsoft, Redmond, WA), and the formal calculations were produced in QC Expert software 3.1 (Trilobyte, Pardubice, Czech Republic) and in IBM SPSS version 17.0 (Chicago, IL).

Results

Demographic characteristics and admission laboratory data of the patients

During September–December of 2012, a total of 121 cases of methanol poisoning occurred in the Czech Republic: 20 patients died outside hospital before treatment initiation and 101 patients were treated in hospital. Of them, in 28 patients, more than three venous blood serum samples were collected consecutively on admission and during hospitalization and stored for LT concentration measurement. These 28 patients were included in the study as the group with acute methanol poisoning (Group I). Other possible causes of elevation of LT concentrations in peripheral blood serum (as bronchial asthma, chronic obstructive pulmonary disease, or other respiratory diseases, chronic neurodegenerative diseases, systemic inflammatory diseases, skin diseases, gastrointestinal diseases, and acute and chronic infectious diseases) were excluded based on medical documentation, anamnesis, medical examination (chest radiographs and others), and laboratory parameters on admission and during hospitalization (complete blood counts and white blood cell differential, sedimentation, C-reactive protein, amylase, and others).

The mean number of blood serum samples was 12 ± 2 per patient and the mean time of observation was 88 ± 20 h. The mean age of the patients in Group I was 54.2 ± 5.2 years, and 9/28 (32%) of patients were females. In the analyzed group, six patients died during hospitalization (cerebral edema confirmed by CT in four cases, cardiac failure in one case, and liver failure in one case) and 22 (79%) patients survived methanol poisoning. Of these 22 survivors, 16 (73%) patients were included in the follow-up clinical examination program and contact was lost with six patients. The data on health sequelae of poisoning in these six patients were collected from discharge reports.

All 80/101 patients who survived acute methanol poisoning during the mass poisoning outbreak in 2012 were invited to participate in the clinical follow-up study of long-term visual and CNS sequelae of poisoning. Of the 50 survivors who agreed with follow-up clinical examination, in 36 (72%) patients control LT concentration in blood serum was measured 2 years after discharge from hospital. These 36 patients constituted a follow-up group for the present study (Group II). This follow-up group consisted of 12 survivors with available results of acute serum LTs measurements (from Group I) and of 24 patients recruited for the clinical follow-up program without acute LTs measurements.

The mean age of the patients in the follow-up group (Group II) did not differ significantly from the mean age of the patients in Group I (47.6 ± 4.5 years; p > 0.05), but the proportion of females in the follow-up group was lower (6/36; 17%; p = 0.001). No patients in the follow-up group had bronchial asthma, chronic obstructive pulmonary disease, or other respiratory diseases, chronic neurodegenerative diseases, systemic diseases, skin diseases, gastrointestinal diseases, and acute and chronic inflammatory and infectious diseases. Hyperlipidemia was diagnosed in 21 patients, alcoholic hepatopathy in 12 patients, arterial hypertension in three patients, diabetes mellitus type II in two patients, and hypothyroidism in one patient in the follow-up group. One patient had brain stroke and one patient had epilepsy in anamnesis.

The admission laboratory biochemical and toxicological parameters measured upon hospitalization with acute methanol poisoning in patients with acute LTs measurements (Group I) and in patients with the follow-up LTs measurements (Group II) are shown in Table 1. There was no difference between the two groups in the time of presentation to hospital and in hospital admission laboratory parameters. The patients in both groups were severely poisoned with high anion gap, base deficit, serum methanol, formic acid, and lactate concentrations, and low bicarbonate. The patients in Group I had insignificantly lower arterial blood pH and bicarbonate than the controls due to the presence of the patients who died during hospitalization.

Table 1. Admission laboratory data in the patients with acute serum LT measurements (Group I) and in the follow-up group of survivors 2 years after discharge (Group II).

Groups	MetOH, mg/L	EtOH, mg/L	Formic acid, mg/L	pH	HCO3^-(mM)	Lactate, (mM)	BE, (mM)	Glucose, (mM)	Creatinine, (μM)	AG (mM)	Time, h
Group I  (n = 28)	1220 ± 420	160 ± 130	590 ± 140	7.06 ± 0.10	9.1 ± 2.7	5.4 ± 2.0	−18.2 ± 5.6	9.7 ± 2.0	95.0 ± 17.0	29.7 ± 4.0	38.4 ± 8.3
Group II  (n = 36)	1340 ± 440	490 ± 340	500 ± 180	7.19 ± 0.27	12.4 ± 2.6	3.6 ± 1.6	−15.2 ± 3.9	7.6 ± 1.2	93.0 ± 13.0	27.1 ± 3.9	33.5 ± 8.0
pI/Control	0.706	0.078	0.419	0.392	0.088	0.184	0.355	0.079	0.837	0.355	0.408

LTs: leukotrienes; MetOH: serum methanol on admission; EtOH: serum ethanol concentration on admission; Formic acid: serum formic acid on admission; BE: base excess; AG: anion gap; pH: arterial blood pH; HCO₃^-: bicarbonate concentration; Lactate: serum lactate on admission; Glucose: serum glucose on admission; Creatinine: serum creatinine on admission; Time: time from methanol ingestion to hospital admission and treatment. p < 0.05 was considered as significant.

Acute serum LTs concentration during the treatment and the outcome of methanol poisoning

The maximum (C_max) and mean (C_mean) serum concentrations of cys-LTs and LTB4 measured during hospitalization were significantly higher than the follow-up LT concentrations measured 2 years after discharge (Figure 1). The difference between two groups remained significant after exclusion of 6 patients who died during hospitalization (p < 0.001 for all measured LTs).

Figure 1. Box-and-whisker plots of serum LT concentrations measured during hospitalization and in the survivors 2 years after discharge (Follow-up). Acute Cmax – maximum serum LT concentrations measured during hospitalization; Acute Cmean – mean serum LT concentrations during the observation period with acute methanol poisoning. Mean (dotted line), standard error of the mean (SEM, box), and 95%CI of the mean (whiskers) are presented. ***p < 0.001.

Within the group of patients with serial acute LTs measurements performed during hospitalization with methanol poisoning (Group I), the maximum and mean concentrations of LTC4 and LTD4, as well as the maximum concentration of LTE4, but not LTB4 were significantly higher in survivors than in those who died (Table 2). After elimination of one outlier who died due to liver failure caused by alcoholic cirrhosis 2 months after admission, the maximum and mean acute concentrations of all four LTs in patients who died were significantly lower than in survivors. The time of observation in survivors and in those who died did not differ (86 ± 21 versus 98 ± 73 h, respectively; p = 0.639). Acute maximum and mean serum concentrations of LTB4 and LTE4, but not LTC4 and LTD4, were significantly higher in survivors without sequelae than in those who survived with long-term visual and/or CNS damage (Table 2).

Table 2. Serum LT concentrations measured during hospitalization (Group I) and in the follow-up group of survivors (Group II) 2 years after discharge (means ± CI95%)^b.

Patients	LTC4, Cmax, pg/mL	LTC4, Cmean, pg/mL	LTD4, Cmax, pg/mL	LTD4, Cmean, pg/mL	LTE4, Cmax, pg/mL	LTE4, Cmean, pg/mL	LTB4, Cmax, pg/mL	LTB4, Cmean, pg/mL
Group I (n = 28)	80.7 ± 5.6	67.0 ± 3.8	51.0 ± 6.6	35.4 ± 3.2	64.2 ± 6.0	49.8 ± 4.5	59.8 ± 6.2	44.4 ± 4.7
Group II (n = 36)	47.9 ± 4.5	47.9 ± 4.5	23.1 ± 2.1	23.1 ± 2.1	26.2 ± 3.9	26.2 ± 3.9	27.2 ± 1.4	27.2 ± 1.4
PI/Control	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
Survived (n = 22)	83.9 ± 6.2	69.3 ± 4.0	54.2 ± 8.0	36.9 ± 3.8	66.8 ± 6.7	51.6 ± 4.6	61.0 ± 6.9	45.0 ± 5.2
Died (n = 6)	68.8 ± 8.6	58.6 ± 7.6	39.5 ± 2.9	29.9 ± 2.3	55.0 ± 15.0	43.0 ± 16.0	55.0 ± 19.0	42.0 ± 15.0
Died^a (n = 5)	66.0 ± 6.1	55.8 ± 2.6	39.6 ± 3.9	29.7 ± 3.0	49.2 ± 7.5	37.4 ± 6.1	47.8 ± 2.2	36.5 ± 3.3
p survived/died	0.023	0.017	0.002	0.003	0.004	0.0140	0.455	0.646
P^asurvived/died	<0.001	<0.001	0.002	0.003	<0.001	0.008	0.001	0.007
No sequelae (n = 11)	88.2 ± 9.2	71.4 ± 5.7	61.0 ± 14.0	39.8 ± 6.5	73.5 ± 9.8	56.5 ± 7.0	69.0 ± 11.0	51.0 ± 9.3
Sequelae (n = 11)	79.6 ± 9.3	67.2 ± 6.4	47.7 ± 7.9	34.0 ± 4.3	60.2 ± 8.7	46.6 ± 5.3	52.6 ± 6.4	39.0 ± 3.0
p no sequelae/sequelae	0.178	0.315	0.114	0.126	0.044	0.027	0.011	0.022

LT: leukotrienes; C_max: maximum serum LT concentrations measured during hospitalization; C_mean: mean serum LT concentrations during the observation period with acute methanol poisoning; No sequelae: the patients in Group I survived without health sequelae of acute methanol poisoning; Sequelae: the patients in Group I survived with long-term visual and/or CNS sequelae of acute methanol poisoning. p < 0.05 (bold numbers) was considered as significant.

^a After elimination of the outlier died due to liver failure caused by alcoholic cirrhosis 2 months after admission.

^b Normal reference ranges in blood serum of healthy human adults for LTC4: 38.0 ± 19.0 pg/mL, for LTD4: 20.9 ± 2.0 pg/mL, for LTE4: 13.6 ± 6.6 pg/mL, and for LTB4: 32.6 ± 2.1 pg/mL [57].

The dynamics of acute serum LT concentration changes during the observation period is shown in Figure 2. The concentration of all four LTs increased during the first 4–5 days of hospitalization and achieved its peak approximately 100–120 h after admission. After the peak, acute LT concentrations decreased during the following days of hospitalization. The approximate rate of increase in acute serum concentration was 0.4–0.5  pg/mL/h for all measured LTs.

Figure 2. Dynamics of acute serum LT concentrations changes during the observation period in the patients hospitalized with acute methanol poisoning.

The acute serum LT concentrations correlated with severity of metabolic acidosis characterized by base excess (r = 0.470, 0.451, and 0.464, all p < 0.05, for LTB4, LTC4, LTD4, and r = 0.502, p < 0.01, for LTE4, respectively) and anion gap (r = −0.507, −0.511, both p < 0.01, for LTC4, LTE4, and r = −0.478, p < 0.05, for LTB4, respectively). The patients with more severe acidemia (higher base deficit, anion gap, lactate, lower arterial blood pH) and therefore more severely poisoned had significantly lower acute serum LT concentrations than those with minor acidemia.

No association was found between acute LT concentration and age, gender, time to hospital presentation, time of observation in this study, Glasgow coma scale, serum methanol, formic acid, glucose concentration on admission, type of antidote administered in hospital (ethanol or fomepizole), and folate substitution (all p > 0.05). The patients administered pre-hospital ethanol as a “first aid antidote” before admission and initiation of hospital treatment had higher serum LTC4 than those without prehospital ethanol (r = 0.509, p < 0.01). Positive serum ethanol concentration on admission was associated with higher acute LTC4 concentration (r = 0.404, p < 0.05). Application of intermittent hemodialysis was associated with higher acute serum cys-LT concentration than in the patients treated with CRRT (r = 0.516, 0.492, both p < 0.01, for LTC4, LTE4, and r = 0.418, p < 0.05, for LTD4, respectively).

Of 28 patients with acute serum LTs measurement, 11 patients survived without sequelae of poisoning, two patients survived with visual sequelae, further two patients survived with CNS sequelae, seven patients had both visual and CNS sequelae, and six patients died in hospital. Significant association was present between acute serum concentration of all four LTs and the outcome of poisoning categorized as “no sequelae”, “visual sequelae”, “CNS sequelae”, “visual and CNS sequelae”, and “death”. This association was stronger for cys-LTs than for LTB4 (r = −0.550, −0.510, and −0.512, all p < 0.01, for LTC4, LTD4, LTE4, and r = −0.417, p < 0.05, for LTB4, respectively). The patients with better outcome had higher acute serum LT concentration during hospitalization than those with poor outcome (death or visual/CNS sequelae).

Control serum LTs concentration and the results of clinical follow-up examination

In the control group, the follow-up clinical examination 3–8 months and 2 years after discharge did not reveal any visual or CNS sequelae of methanol poisoning in 17/36 (47%) of the patients. In the other 19 patients, visual sequelae were diagnosed in five cases, CNS sequelae in five cases, and both visual and CNS sequelae were found in nine cases. The patients with visual sequelae had abnormal VEP and RNFL measurements with concurrent pathologic findings on fundus, perimeter, visual acuity, color vision, and contrast sensitivity. In 14 patients with MRI signs of CNS sequelae, symmetrical necrosis of putamen was registered in eight cases, necrotic lesions of nucleus pallidus in five cases, subcortical hemorrhagic necrosis of white matter in four cases; necrosis of nucleus dentatus, nucleus lentiformis, and pons were found each in one case. Control serum concentration of cys-LTs and LTB4 measured 2 years after discharge from hospital in survivors with and without long-term sequelae of poisoning did not differ (Table 3).

Table 3. Follow-up serum LT concentrations measured 2 years after discharge (Group II) in survivors without sequelae versus survivors with long-term visual and/or CNS sequelae of poisoning (n = 36; means ± CI95%)^a.

Patients	LTC4, pg/mL	LTD4, pg/mL	LTE4, pg/mL	LTB4, pg/mL
No sequelae (n = 17)	46.4 ± 8.5	23.6 ± 3.3	25.2 ± 6.4	27.6 ± 2.1
Sequelae (n = 19)	49.4 ± 4.6	22.5 ± 2.9	27.1 ± 5.4	26.8 ± 2.1
p	0.528	0.604	0.646	0.587

LT: leukotrienes; No sequelae: the patients in the follow-up Group II survived without health sequelae of acute methanol poisoning; Sequelae: the patients in the follow-up Group II survived with long-term visual and/or CNS sequelae of acute methanol poisoning. p < 0.05 was considered as significant.

^a Published normal reference ranges in blood serum of healthy human adults for LTC4: 38.0 ± 19.0 pg/mL, for LTD4: 20.9 ± 2.0 pg/mL, for LTE4: 13.6 ± 6.6 pg/mL, and for LTB4: 32.6 ± 2.1 pg/mL [57].

The decrease in serum LTs Cmax measured during hospitalization compared to the follow-up LT concentrations measured 2 years after discharge in the same patients is illustrated in Figure 3. The mean decrease in LT concentrations in these patients was: LTC4 by 30.9 ± 9.0  pg/mL (or by 38.0 ± 11.0%), LTD4 by 26.3 ± 8.6  pg/mL (51.0 ± 12.0%), LTE4 by 37.3 ± 6.4  pg/mL (59.7 ± 9.3%), and LTB4 by 32.0 ± 8.8  pg/mL (53.0 ± 8.5%). The follow-up concentrations of LTC4, LTD4, and LTB4 did not correlate with acute LT concentrations measured in the same patients (all p > 0.05). Only the follow-up LTE4 concentration correlated with acute LTE4 concentration (r = 0.692; p = 0.013).

Figure 3. Decrease of serum LT concentrations measured during hospitalization (“Acute”) versus LT concentrations measured 2 years after discharge in the same patients (“Follow-up”).

The results of the follow-up LTs measurement in 36 patients 2 years after discharge did not correlate with the demographic characteristics of the patients (age, gender), clinical and laboratory parameter measured on admission and during hospitalization, treatment modalities, and the outcome of poisoning. No correlation of the control LT concentration was found with laboratory parameters measured within the follow-up clinical examinations (glycemia, glycohemoglobin, liver enzymes, urea, creatinine, uric acid, CDT, TSH, vitamins B₁₂ and B₁, and ethyl glucuronide in urine), as well as with the results of VEP and RNFL measurements, and MRI examination of the brain.

Discussion

Our findings suggest that leukotriene-mediated neuroinflammation may play an important role in the mechanisms of toxic brain damage in acute methanol poisoning in humans. The patients with acute methanol poisoning had significantly higher concentrations of cys-LTs and LTB4 in peripheral blood serum than the survivors of poisoning examined 2 years after discharge. During hospitalization, the patients who survived poisoning had higher acute concentration of all four measured LTs than those who died. In survivors, the patients with poor outcome (visual and CNS sequelae) had lower acute serum LT concentration than those who survived without health sequelae, but higher than those who died. Significant association between acute serum LT concentration and the outcome of poisoning may indicate the neuroprotective effect of a moderate increase in LT concentration observed in patients with methanol poisoning. The LT concentration peaked 4–5 days after admission, when formic acid had already been eliminated and acidemia fully corrected. The absence of association between acute and follow-up LT concentrations, along with the absence of difference in the follow-up LT concentration between the patients who survived with and without visual/CNS sequelae, indicated that acute elevation of LT concentration was moderate, adaptive, transitory, and was not followed by chronic leukotriene-mediated neuroinflammation in survivors during the two-year period after discharge from hospital.

The pathophysiology of CNS damage in acute methanol poisoning has a complex and not fully understood mechanism. Formic acid, the main toxic metabolite of methanol, induces cellular toxicity through inhibition of cytochrome c oxidase, which impairs oxygen utilization, causing a shift from aerobic to anaerobic metabolism [14,15]. If ADH is blocked by antidote (ethanol or fomepizole), formic acid is effectively eliminated by hemodialysis with a half-life of 1.6–3.6 h and acidemia is corrected during the first hours after hospital treatment initiation [33,34]. In addition to its primary cytotoxic effect, formic acid induces secondary effects including ischemia, edema, BBB disruption, hemorrhages, reactive oxidative damage, axonal demyelination, neuronal degeneration, and cell death.

Neuroinflammation as a result of endogenous immune activation in the brain is a universal response of the CNS to the injury aimed at limiting damage, restoring homeostasis, protecting neurons, and promoting tissue recovery and repair [18–21]. Activated by primary traumatic or non-traumatic brain injury, microglia produces several types of inflammatory mediators including LTA4 and LTB4 [37,38]. Reactive astrocytes produce LTB4 and all three cys-LTs and form a physical barrier between the lesioned and the healthy tissues, demarcating them to promote recovery and prevent further damage [39,40].

In the healthy brain, LT concentrations are very low or absent [41]. However, all LTs are markedly elevated and LT receptors Cys-LT1 and Cys-LT2 are up-regulated in the brain after TBI or focal ischemia [19–23,39]. Experimental studies demonstrated that brain-specific proteins (glial fibrillary acidic protein, ubiquitin carboxy-terminal hydrolase L1, and others) are able to reach peripheral circulation through a disrupted BBB and via the glymphatic system [25,28].

There are no published data on the role of LTs in the non-traumatic brain injury caused by acute methanol poisoning. In humans, LT concentrations were elevated in exhaled breath condensate, peripheral blood, and urine of patients with chronic intoxication with neurotoxic 2,3,7,8-tetrachloro-dibenzo-p-dioxin, and healthy subjects occupationally exposed to TiO₂ nanoparticles [42,43]. Further, there is clear evidence that acute organophosphorus compounds intoxication is associated with neuroinflammatory response (review by Banks and Leins) [44]. In experimental studies, single mild impact to the brain after closed head injury without BBB disruption initiated prolonged endogenous neuroinflammatory response [25].

In our study, the maximum and mean serum concentrations of all four LTs measured in patients hospitalized with acute methanol poisoning were significantly higher than in the follow-up group. Two years after discharge, serum concentrations of LTs measured in the same patients was 40–60% lower than their acute concentrations. These results suggest that leukotriene-mediated neuroinflammation may play an important role in the mechanisms of nontraumatic brain damage in acute methanol poisoning.

The dynamics of LT concentration changes during hospitalization demonstrated an almost linear increase with the peaks on the fourth–fifth day of observation followed by a slow decrease during the second week after admission. In experimental TBI, LTs production was very rapid, peaking at 1–3 h after injury [41,45]. In our study, no peak concentration was detected during the first day of observation. This fact can be explained by the different mechanisms of brain damage. In experimental TBI, the mechanical force can directly shear blood vessels and axons, damage neurons and glia, leading to intracerebral bleeding, brain tissue compression, lacerations, and contusions with quick activation of several biochemical cascades including neuroinflammation [46,47]. In non-traumatic brain damage caused by accumulating formic acid, the secondary effects evolve gradually through several stages during approximately 1 week after admission, as was demonstrated by the series of brain CT/MRI examinations during hospitalization in patients with acute methanol poisoning [9]. Our results correspond with the results of experimental studies demonstrating that neuroinflammatory responses persist for several weeks with zenith LT concentrations around day seven after brain injury [21,48,49].

The severity of metabolic acidosis on admission is an important prognostic parameter in acute methanol poisoning [50]. There is a strong correlation between the degree of acidemia and the probability of long-term visual and CNS sequelae of poisoning in survivors [2–5,11]. In our study, acute serum LT concentration negatively correlated with severity of acidemia: in more acidemic patients, the acute concentration of LTs was lower. These results might indicate that the leukotriene-mediated response of the innate immune system in the brain was inhibited in the most severely poisoned patients by formic acid and acidemia. Formic acid is a weak acid (pK_a = 3.78) and a pH drop of 0.3 would mean doubling the undissociated formic acid levels, hence a significant increase in toxicity. Only undissociated formic acid is able to cross the membranes and inhibit cytochrome c oxidase in mitochondria of neurons, microglia, and astrocytes.

Lower acute concentrations of all four LTs were associated with poor outcome of poisoning (death or survival with visual/CNS sequelae). The patients who died had the lowest concentration of cys-LTs and, after elimination of the outlier who died due to alcoholic cirrhosis 2 months after admission, of LTB4 as well. In survivors, LT concentrations were higher in patients who survived without sequelae compared to the patients who survived with visual and CNS sequelae of poisoning. This association suggests that the acute elevation of LT concentrations observed in patients with methanol poisoning had rather a neuroprotective than a maladaptive character.

Interestingly, protective positive serum ethanol concentration on admission was positively associated with acute LTC4 concentration. In animal models of cerebral, renal, liver, and cardiac ischemia, alcohol exposure was shown to reduce ischemia reperfusion injury and prevent post-ischemic adhesive interactions between leukocytes and endothelial cells which can lead to organ dysfunction and death [51,52]. The patients who received pre-hospital ethanol as a “first aid antidote” before admission to hospital had higher serum LTC4 concentration and had better outcome of poisoning than those with negative serum ethanol on admission, as was shown in our earlier studies [53,54]. Finally, the patients treated with IHD had higher concentration of cys-LTs than those treated with CRRT. This fact can be explained by more rapid elimination of formic acid and correction of acidemia in patients treated with IHD [33,34].

Several experimental studies demonstrated the protective effect of leukotriene receptor antagonists and 5-LO activating protein (FLAP) inhibitors blocking the synthesis of all four LTs administered before TBI on injury-related outcomes [21,41,45]. Positive effects of inhibition of LT synthesis in these studies can be explained by a different mechanism, severity, and extent of experimental TBI and non-traumatic brain injury caused by methanol poisoning. In TBI, focal or diffuse mechanical damage to the cerebral tissue causes an uncontrolled, excessive release of excitatory neurotransmitters, rapidly triggering a cascade of events called excitotoxicity [55]. Vascular damage in TBI leads to the infiltration of peripheral blood immune cells into brain tissue, which produce lipid inflammatory mediators, chemokines, inflammatory cytokines, and oxidative molecules. In these conditions, synthesis of LT and other pro-inflammatory lipid mediators by activated microglia and astrocytes may be excessive, and therefore maladaptive. Bi-directional relationship between microglia and astrocytes that allow them to act in concert to regulate neuroinflammatory process may be dysregulated, which may exacerbate tissue damage and contribute to the detriment of TBI [48,49]. Administration of FLAP inhibitors in this case may interrupt a vicious self-perpetuating cycle of microglia and astrocyte activation and the resolve of neuroinflammatory response [24,25]. In acute methanol poisoning, the increase in LT synthesis was adaptive, transient, and was apparently inhibited by formic acid and acidemia in the most severely poisoned patients. The difference between acute and the follow-up LT concentrations in survivors was about 40–60%, therefore, relatively moderate.

Prolonged excessive activation of microglia in brain injury can drive chronic neuroinflammation and neurodegeneration. Activated microglia and reactive astrocytes are supposed to play an important role in the inflammation-mediated mechanisms of chronic neurodegenerative diseases as Alzheimer’s disease [56]. Inability of the brain to turn off or resolve neuroinflammation can lead to progressive axonal injury and chronic neurodegenerative processes.

In our study, no cases of persisting elevation of LT concentration 2 years after discharge from hospital were registered. The difference in serum LT concentration between the survivors with and without visual/CNS sequelae of poisoning in the follow-up group was insignificant. The follow-up serum concentration of LTs correlated neither with acute parameters of methanol poisoning, nor with laboratory and clinical parameters measured within the follow-up examination including the results of VEP, OCT-RNFL, and MRI examinations. The concentration of LTs in the follow-up group corresponded to the normal reference ranges in blood serum of healthy adults [57]. The results of our study indicated that methanol-induced acute leukotriene-mediated neuroinflammation resolved in survivors without the shift to chronic inflammation.

Limitations of the study

There were several limitations to the design of this study. The relatively limited sample size of the group with acute methanol poisoning could lead to insufficient power of the study in determining the association of LT concentration with certain laboratory and clinical parameters measured during hospitalization. The study was not controlled with regard to the co-morbidity of the patients hospitalized with acute methanol poisoning who were included in the study. Nevertheless, the study population was relatively young; the proportion of patients with co-morbidities in the follow-up group was low, and the parameters studied during the follow-up clinical examination allowed us to exclude these co-morbidities as possible causes of increased serum LT concentration.

The study was not controlled for the time to hospital presentation (time from methanol ingestion to the start of hospital treatment), time of blood collection for biochemical and toxicological laboratory analysis (the laboratory parameters measured from the first blood sample collected in a hospital were assumed as the parameters measured on admission), and treatment modality (choice of antidote and mode of dialysis, alkalinization, and folate substitution). These confounders may affect the outcome of treatment.

The aim of the study was to compare serum LT concentrations in patients with acute methanol poisoning and in survivors 2 years after discharge, to test the association with long-term changes in functional state of the optic nerve and morphological state of the retina and the brain. We did not study possible effects of different treatment modalities on the outcomes and sensitivity of serum LT concentration as a prognostic parameter of visual and CNS sequelae after acute methanol poisoning. Finally, a substantial number of survivors of methanol poisoning did not participate in the follow-up clinical examination; therefore, selection bias is possible, with fewer severely affected patients participating in follow-up. Nevertheless, the demographic and admission laboratory parameters characterizing the severity of poisoning did not differ between groups.

Despite the limitations, this is the first longitudinal study, which provides the data on acute and control serum LT concentrations measured in the cohort of methanol-poisoned patients hospitalized during a mass poisoning outbreak and in the survivors 2 years after discharge. The study demonstrates the association of acute serum LT concentrations with admission laboratory parameters of severity of poisoning, outcome of treatment, and long-term health sequelae. In the cohort of survivors, the follow-up clinical examination was performed two times during 2-year period after discharge to provide detailed information on the character of visual and CNS damage. The follow-up examination of survivors was performed in one hospital using a defined protocol and advanced technology to identify and objectively characterize the clinical lesions of retina, the optic nerve, and the brain. All patients were examined according to the same standardized protocol including complete ophthalmologic and neurological examinations, as well as biochemical and toxicological tests to limit influence of other confounders.

Disclosure statement

The authors report no conflict of interest. The authors alone are responsible for the content and writing of this paper. The manuscript has been read and approved by all authors. The authors certify that the submission (aside from an abstract) is not under review at any other publication. The authors certify that the authors have no other submissions and previous reports that might be regarded as overlapping with the current work.

Additional information

Funding

This work was supported with the Project 16-27075A of AZV VES 2016 of Ministry of Health, and the Projects PROGRES Q25 and Q29, 1st Faculty of Medicine, Charles University in Prague.