Molecular Hydrogen Therapy in Aneurysmal SAH With RA and Newly-diagnosed SLE, Complicated With Acute Ischemic Infarction: A Case Report of Improved Immune Markers Including Tr1 Cells, Breg Cells and TIM3 Expression on Tc Cells

Abstract

Background/Aim

Most nontraumatic subarachnoid hemorrhages (SAHs) are caused by ruptured saccular aneurysms, often resulting in a devastating clinical event characterized by high mortality and significant morbidity among survivors. Numerous studies have confirmed the neuroprotective effects of the molecular hydrogen due to its unique biological properties.

Case Report

We present the case of a 44-year-old female with aneurysmal SAH with rheumatoid arthritis (RA) and newly diagnosed systemic lupus erythematosus (SLE), complicated by acute ischemic infarction. Despite surgical, pharmacological and non-pharmacological interventions, including embolization of the aneurysm, immunosuppressant, non-vitamin K antagonist oral anticoagulant (NOAC), and plasmapheresis, loss of consciousness continued. The patient began daily treatment with hydrogen capsules, resulting in increased in Treg cells, Breg cells, increased TIM3+ expression on Tc cells, and the conversion of anti-dsDNA from positive to negative. Her clinical symptoms stabilized without adverse effects.

Conclusion

This case highlights the potential benefits of molecular hydrogen therapy in managing aneurysmal SAH with underlying autoimmune disease, warranting further research.

Most nontraumatic subarachnoid hemorrhages (SAHs) are caused by ruptured saccular aneurysms. This condition is often a devastating, with substantial mortality and high morbidity among survivors. Generally, one-third of patients who experience an aneurysmal subarachnoid hemorrhage (SAH) will recover well, one-third will survive with a disability or stroke, and one-third will die. Cumulative case fatality rates after SAH are 25-30% on the first day, 40-45% within the first week, 50-60% after the first month, and 55-60%, 65% and 65-70% at 6, 12 and 60 months respectively. Additionally, 12% of patients die before receiving medical attention (1).

Recent studies have demonstrated that hydrogen therapy exerts its effects through antioxidant, anti-inflammatory, and anti-apoptotic mechanisms. Molecular hydrogen (H₂) can readily diffuse across cell membranes, reducing excessive reactive oxygen species (ROS) and modulating various cellular signaling pathways. This leads to the protection of mitochondria, enhancement of adenosine triphosphate (ATP) production, and regulation of cell death processes, such as apoptosis and autophagy (2). Applications of hydrogen therapy include treatment for cardiovascular diseases, ischemia-reperfusion injuries, brain injuries, neurodegenerative diseases, respiratory disorders and even coronavirus disease 2019 (COVID-19) (2-4).

We report a case of SAH with rheumatoid arthritis (RA) and newly diagnosed systemic lupus erythematosus (SLE), further complicated by an acute ischemic infarction. Despite various surgical, pharmacological and non-pharmacological interventions, the patient’s loss of consciousness persisted. However, following the administration of hydrogen capsules, the patient’s neurological condition gradually improved. Additionally, we analyzed immune cell phenotypes before and after treatment to characterize the responses and assess the effectiveness of hydrogen-assisted therapy. This study was approved by the Institutional Review Board (IRB) of Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, and complied with relevant guidelines (IRB: B202105106, approval date: 16 January 2024). Written informed consent was obtained from the patient (Consent No. B202105106-24). The study adhered to the ethical standards of the institution as well as the 1964 Helsinki Declaration and its subsequent amendments or equivalent ethical standards.

Case Report

A 44-year-old woman presented to the emergency department with a one-week history of neck pain and stiffness, accompanied by the sudden onset of an extremely painful headache. Approximately 15 years prior to the current evaluation, the patient was diagnosed with RA and possible deep vein thrombosis. Her RA was managed with prednisolone, leflunomide, hydroxychloroquine, sulfasalazine and tofacitinib. Rivaroxaban was prescribed for the possible deep vein thrombosis.

One week before the current admission, the patient experienced neck pain with stiffness and chest pain. She visited our emergency department, where a chest X-ray revealed left lower lung infiltration with left pleural effusion, leading to a diagnosis of bronchopneumonia. She was treated with antibiotics and discharged the following day. However, neck pain with stiffness persisted, and she subsequently developed a sudden, extremely strong headache. As a result, she returned to the emergency department of this hospital.

The patient’s temperature was 37.2˚C, heart rate was 87 beats per minute, blood pressure was 161/84 mm Hg, and respiratory rate was 25 breaths per minute. Her oxygen saturation was 95% while breathing ambient air. On examination, she appeared acutely ill and weak. Her Glasgow Coma Scale (GCS) score was E3M6V4. Both Kernig and Brudzinski signs were negative, no other neurologic deficits were identified. Brain computed tomographic (CT) and CT angiography (CTA) were performed, revealing diffuse acute subarachnoid hemorrhage on CT (Figure 1A) and no evidence of large vessel occlusion, aneurysm or vascular malformation (Figure 1B). Consequently, the patient was admitted to our ICU for further evaluation and management.

Figure 1.

Sequential imaging and endovascular treatment of a left anterior communicating artery aneurysm. (A) Brain computed tomographic (CT) on December 25, 2023, showing diffuse acute subarachnoid hemorrhage. (B) Brain angiography (CTA) on December 25, 2023, indicating no evidence of large vessel occlusion, aneurysm or vascular malformation. (C) Internal carotid angiography on December 26, 2023, revealing a thin-tubular aneurysm arising from the left anterior communicating artery. (D) 3D angiography on December 26, 2023, showing the aneurysm in detail, measuring 2 mm in length and 0.75 mm in diameter, arising from the left anterior communicating artery. (E) Y-stent-assisted coiling embolization of the aneurysm was performed on December 29, 2023. Post-embolization angiography of the left internal carotid artery demonstrated patency of the Y-stent with contrast filling within the aneurysmal pseudo-sac.

After admission, internal carotid angiography was performed, revealing a thin-tubular shape aneurysm measuring 2 mm in length and 0.75 mm in diameter arising from the left side of the anterior communicating artery (Figure 1C and D). Consequently, the patient underwent Y-stent-assisted coiling embolization of the aneurysm four days later (Figure 1E). However, her consciousness deteriorated following the procedure. As there was no improvement in consciousness after 2 weeks of observation, a brain magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA) were conducted. These revealed diffusion restriction in the bilateral cerebral white matter, left insula, and left parieto-occipital lobe, indicative of acute ischemic infarction (Figure 2A).

Figure 2.

Imaging follow-up after Y-stent-assisted coiling embolization of a left anterior communicating artery aneurysm. (A) Brain and magnetic resonance angiography (MRA) on January 11, 2024, showing diffusion-restriction in bilateral cerebral white matter, the left insula, and the left parieto-occipital lobe, indicative of acute ischemic infarction. (B) Follow-up cerebral angiography on March 12, 2024 (two months after Y-stent-assisted coiling embolization of the aneurysm), showing regression of the previously embolized aneurysm. (C) Brain computed tomography (CT) on March 12, 2024, indicating no new hemorrhagic lesions.

Interestingly, the brain MRI and MRA lesions were not compatible with the location of aneurysm. Upon admission, the rheumatology and immunology physician ordered a series of examinations, including various autoimmune markers. Laboratory data on the day of admission revealed a positive antinuclear antibody (ANA) titer of 1:1,280 and lupus anticoagulant. One week later, all tests for anti-dsDNA, anti-Sm antibody, anti-RNP antibody and anti-histone antibody returned positive. A low level of C4 was also noted. Based on the clinical features of joint involvement and the laboratory findings, SLE was confirmed according to the 2019 EULAR/ACR classification criteria for SLE (5). The physician also tested for markers of antiphospholipid syndrome (APS). However, the presence of lupus anticoagulant alone did not satisfy the laboratory domains of the 2023 ACR/EULAR APS classification criteria (6).

During hospitalization, the patient received supportive care, empiric antibiotics, and anticoagulation therapy. Following her new diagnosis with SLE, she underwent plasmapheresis three times. Additionally, after discussion and agreement with the patient’s family, molecular hydrogen therapy (1 capsule/day) was initiated on January 16, 2024. The hydrogen capsules (PURE HYDROGEN) were purchased from HoHo Biotech Co., Ltd. (Taipei, Taiwan, ROC). Each capsule contained 170 mg of hydrogen-rich coral calcium, equivalent to 1.7×10²¹ molecules of hydrogen, which corresponds to 24 cups of water with 1,200 ppb of hydrogen or 0.6 mM of hydrogen per 200 ml of water. Before the initiation of hydrogen capsule therapy, the muscle strength in all four limbs was rated at 1. By the first month of hydrogen therapy, the patient had successfully weaned off the ventilator. Muscle strength improved in the left upper limb from 1 to 3, and left lower limb from 2 to 3. Additionally, anti-dsDNA decreased from positive to negative, the GCS score improved from E3M2VT to E4M6VT. As of the sixth month, muscle strength had further improved to 5 in the left upper limb, 4 in the left lower limb, 3 in the right upper limb, and 2 in the right lower limb. Follow-up imaging showed regression of the aneurysm and no new hemorrhagic lesions (Figure 2B and C). By the sixth month of hydrogen therapy, the patient was able to produce and recognize some simple words. Importantly, since the patient’s SAH required discontinuation of tofacitinib upon admission, we did not resume the medication during her hospitalization, and her RA remained well-controlled (Figure 3).

Figure 3.

Disease progression and clinical treatment course. The black line represents overall disease progression. The blue text indicates the patient’s medication. The green text denotes laboratory and imaging examinations and their findings. The orange text describes the patient’s neurological condition.

Flow cytometry and serological examination were utilized for whole-blood analysis to evaluate changes in immune cells and autoantibodies before and after hydrogen therapy. For subsequent whole-blood analysis via flow cytometry, blood samples were processed according to standard procedures involving fluorescent dye preparation and fluorescent antibody reagent kits with dried reagents (Beckman Coulter, Brea, CA, USA). The methods, steps, immunophenotypic analysis, and cell gating were performed according to previously established protocols (7-10). Our analysis of immunophenotypic markers before and after hydrogen therapy revealed an increase in TIM3+ (T cell immunoglobulin and mucin-domain containing-3) cytotoxic T cells (Tc cells), regulatory B (Breg) cells and programmed cell death protein 1 (PD-1) expression on naïve T helper cells (Th cells). Additionally, after 4 months of hydrogen therapy, CD21+, HLADR+, PD-1+ switched memory (SM) B cells and CD127+, activated regulatory T (Treg) cells and T regulatory type 1 (Tr1) cells returned to pre-treatment levels (Figure 4). Among these findings, the most notable was the Tr1 cells, whose levels decreased before hospitalization and then gradually increased as the patient’s condition improved following hydrogen therapy, as shown in Figure 4E. TIM3+ Tc cells and Breg cells also demonstrated favorable responses to hydrogen therapy (Figure 4A-C).

Figure 4.

Immunophenotypic changes before and after molecular hydrogen therapy. Molecular hydrogen therapy (1 capsule/day) was initiated on January 16, 2024. Whole-blood analyses were conducted eight times: prior to therapy on October 17, 2019; April 21, 2022; October 6, 2022; March 23, 2023; and January 8, 2024, and after therapy on January 22, 2024; February 19, 2024; and May 17, 2024. (A-B) Percentage change in TIM3+ expression on Tc cells before and after therapy. (C) Percentage change in Breg cells before and after therapy. (D) Percentage change in PD-1 expression on naïve Th cells before and after therapy. (E) Percentage change in Tr1 cells before and after therapy. (F-H) Percentage change in CD21+, HLADR+, PD-1+ SM B cells before and after therapy. (I) Percentage change in CD127+ Treg cells before and after therapy. (J) Percentage change in activated Treg cells before and after therapy.

Discussion

The pathophysiological changes following SAH are commonly divided into early brain injury (EBI) and delayed brain injury (DBI). EBI is associated with several events, including increased intracranial pressure (ICP), decreased cerebral blood flow, and global cerebral ischemia occurring after SAH. These events can lead to blood-brain barrier disruption, inflammation, brain edema and neuronal cell death (11). DBI, which manifests as focal neurological deficits and/or cognitive impairments, is considered the most significant cause of mortality and morbidity following SAH (12). S100B secretion from astrocytes increases after brain injury, leading to elevated reactive oxygen species (ROS) levels in astrocytes, microglia, and neurons. The increased ROS induce phosphorylation of c-Jun N-terminal kinase (p-JNK) in neurons, triggering apoptosis. Enhanced S100B release transforms astrocytes into reactive astrocytes, characterized by up-regulation of glial fibrillary acidic protein (GFAP) and S100B. Additionally, enhanced p-JNK activates microglia, transforming them into reactive microglia. A study demonstrated that inhalation of 1.3% H2 gas ameliorated delayed brain injury (DBI) by early brain injury (EBI) in an experimental rat model of SAH. The study also observed reduced expression of S100B and p-JNK, which play crucial roles in the pathophysiological changes occurring in EBI (13).

This case study underscores the urgent need for novel interventions to treat cases of aneurysmal SAH in patients with RA and newly diagnosed SLE, complicated by acute ischemic infarction. Despite various interventions, including aneurysm embolization, immunosuppressants, NOAC, and plasmapheresis, the patient’s loss of consciousness persisted, and symptoms remained unrelieved. However, after six months of adjuvant hydrogen capsule therapy, improvements were observed in consciousness, muscle strength, and cognition. Follow-up imaging revealed no new hemorrhagic or ischemic lesions. Additionally, anti-dsDNA antibody levels transitioned from positive to negative following hydrogen treatment. Importantly, tofacitinib was discontinued upon the patient’s admission due to SAH, and it was not resumed during hospitalization. Despite this, RA remained well-controlled during and after hydrogen therapy.

We conducted an analysis of immune cell phenotypes before and after treatment to characterize responses and assessing the effectiveness of hydrogen-assisted therapy (Figure 4). Our results revealed an increase in TIM3+ Tc cells, Breg cells and PD-1 expression on Th cells. Additionally, after four months of hydrogen therapy, CD21+, HLADR+, PD-1+ SM B cells and CD127+, activated Treg cells and Tr1 cells returned to their pretreatment levels. Among these findings, the most notable were the Tr1 cells, whose levels, as shown in the line graph, corresponded with the course of the disease (Decreasing before hospitalization and gradually increasing as the condition improved with hydrogen therapy) (Figure 4E). TIM3+ Tc cells and Breg cells also exhibited positive responses to hydrogen therapy (Figure 4A-C). Among the genes encoding the TIM family, TIM3 has garnered the most attention due to its role in regulating immune responses in autoimmunity and cancer. Notably, the surface expression of TIM3 on T cells is low in patients with ulcerative colitis, multiple sclerosis, RA and psoriasis compared to healthy controls. This reduced expression aligns with the limited regulation of the inflammatory properties of T cells in patients with these autoimmune diseases (14).

Tr1 cells and FOXP3+ Tregs are two major subsets of regulatory CD4+ T cells. Unlike FOXP3+ Tregs, the master transcription regulator for Tr1 cells remains unidentified. Nonetheless, Tr1 cells are generally defined as a specialized subset of CD4+ T cells that are induced in the periphery during antigen exposure under tolerogenic conditions (15). One of the key features of Tr1 cells is their expression of the immunosuppressive cytokine interleukin-10 (IL-10) and transforming growth factor-beta (TGF-β), which can inhibit the function of effector immune cells independently of forkhead box P3 (FOXP3) expression (16). Bregs are also immunosuppressive cells that downregulate immune responses and support immunological tolerance. The suppressive activities of Breg cells and the molecules responsible for these functions have been partially described. These activities include the inhibition of T cell activation, induction of Tregs, the expression of IL-10, IL-35, TGF-β (17). Both Treg and Breg cells are crucial for maintaining immune homeostasis and tolerance, which helps prevent autoimmune reactions and diseases, such as SLE, RA, type 1 diabetes (T1D), multiple sclerosis (MS) (17,18).

In this case, following hydrogen therapy, the patient did not resume tofacitinib, yet RA remained well-control. Additionally, anti-dsDNA levels transitioned from positive to negative. While direct evidence linking hydrogen therapy to the induction or modulation of Tr1 cells, Breg cells, and TIM3 expression is still emerging, hydrogen’s anti-inflammatory and immunomodulatory effects suggest a potential interaction. By reducing oxidative stress and inflammation, hydrogen therapy may foster a more favorable environment for the function of Tr1 and Breg cells, and enhance TIM3 expression, thereby contributing to improved immune tolerance in autoimmune diseases.

Hydrogen exerts protective effects primarily through its anti-oxidation, anti-inflammation, anti-apoptotic properties, as well as by regulating autophagy, and preserving mitochondrial function and the blood-brain barrier (19). As a neuroprotective gas, H2 offers several advantages. It can cross the blood-brain barrier (BBB), penetrate biological membranes, and diffuse into the cytosol and organelles. Additionally, H2 is simple to administer and has neuroprotective effects with minimal side-effects, suggesting its potential as a therapeutic strategy in clinical settings (20).

Conclusion

In conclusion, this case study highlights the potential efficacy of hydrogen-assisted therapy for a patient with aneurysmal SAH with RA and newly diagnosed SLE, complicated by acute ischemic infarction. While hydrogen-assisted therapy appears promising, the limited sample size of this study necessitates further research with a larger cohort and extended follow-up to confirm its efficacy. Additionally, future studies should aim to elucidate the relationship between hydrogen therapy and Tr1 cells, Breg cells as well as TIM3 expression on Tc cells.

Conflicts of Interest

The Authors have no conflicts of interest or competing interests to disclose in relation to this study.

Authors’ Contributions

YTL: Conceptualization, methodology, writing – original draft, writing review and editing. JWL: Conceptualization, methodology, writing original draft, writing – review and editing. YJH: Conceptualization, methodology, project administration, writing original draft, writing – review and editing. SWL: Conceptualization, methodology, writing original draft, writing – review and editing. TYH: Conceptualization, methodology, writing original draft, writing – review and editing. FCL: Conceptualization, investigation, supervision, writing – review and editing.

Acknowledgements

This study was supported by the Undergraduate Research Fellowship, Ministry of Science and Technology (MOST 111-2314-B-016-026), the National Science and Technology Council (NSTC 112-2314-B-016-033; NSTC 113-2314-B-016-052), and Tri-Service General Hospital (TSGH-E-111215; TSGH-E-112218) in Taiwan.