Re: mircocurrent 치료기 - 동맥의 기형을 개선한다 2024 natrue

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Microcurrent wave alleviates mouse intracranial arterial dolichoectasia development

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• Published: 29 March 2024

Microcurrent wave alleviates mouse intracranial arterial dolichoectasia development

• Jae Hee Lee, 
• Huy Duc Vu, 
• Min Hee Park, 
• Phuong Tu Huynh, 
• Sung Won Youn & 
• Dong Rak Kwon 

Scientific Reports volume 14, Article number: 7496 (2024) Cite this article

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Abstract

Intracranial arterial dolichoectasia (IADE) is associated with the interaction of hypertension and inflammation, and microcurrent can be effective in hypertension. Therefore, this study aimed to investigate the therapeutic effect of microcurrent electrical stimulation in a mouse IADE model. This study randomly categorized 20 mice into five groups: group 1-C (healthy control), group 2-D (IADE model), group 3-M + D (microcurrent administration before nephrectomy and until brain surgery), group 4-D + M (microcurrent administration for 4 weeks following brain surgery), and group 5-M (microcurrent administration for 4 weeks). Cerebral artery diameter and thickness and cerebral arterial wall extracellular matrix components were assessed. Among the five groups, group 2-D showed significantly higher cerebral arterial wall diameter (117.79 ± 17.05 µm) and proportion of collagen (42.46 ± 14.12%) and significantly lower arterial wall thickness (9.31 ± 2.26 µm) and proportion of smooth muscle cell (SMC) and elastin in the cerebral arterial wall (SMC: 38.05 ± 10.32%, elastin: 11.11 ± 6.97%). Additionally, group 4-D + M exhibited a non-significantly lower diameter (100.28 ± 25.99 µm) and higher thickness (12.82 ± 5.17 µm). Group 5-M demonstrated no evidence of toxicity in the liver and brain. The pilot study revealed that microcurrent is effective in preventing IADE development, although these beneficial effects warrant further investigation.

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Introduction

Intracranial arterial dolichoectasia (IADE) is a unique arteriopathy distinguished by its elongation, dilation, and tortuosity1,2,3. IADE demonstrated 0.1%–6.5% preval‎ence rates in the general population, which has increased to approximately 12% in patients with stroke4. IADE can frequently be asymptomatic but may present as a serious cerebrovascular disease, including cerebral infarction, cerebral aneurysm, cerebral hemorrhage, or nearby structure compressions5,6,7. Dolichoectasia-induced ischemic stroke involves two main mechanistic factors: mechanical stretching and blocking of penetrating arteries, as well as flow stagnation due to reduced flow associated with arterial dilatation. The pathogenesis of IADE has been investigated, and it can be described as a common end pathway of arterial wall damage that particularly affects the tunica media related to hypertension6,8,9. A previous study1 reported that IADE development is closely connected to hemodynamic stress-induced inflammation and extracellular matrix (ECM) remodeling within the arterial wall.

Currently, IADE is managed primarily based on the patient’s clinical presentation and the disease severity. This approach includes measures, such as blood pressure control, the use of antithrombotic agents, and in some cases, more invasive treatments, including endovascular procedures and surgery10,11. Antithrombotic medications are often recommended as a prophylaxis for preventing secondary ischemic strokes, but patients with dolichoectasia still experience an increased risk of recurrent ischemic strokes3. Surgical interventions for dolichoectatic aneurysms are challenging to approach, and they require intensive planning and caution to avoid iatrogenic rupture during the procedure11. Safe and non-invasive treatment options that consider the underlying pathophysiological factors to be corrected need to be explored because the optimal treatment for IADE still has no consensus.

This study considered microcurrent therapy as the potential strategy to modify the natural course of IADE because microcurrent therapy addressed hypertension and inflammation simultaneously. Microcurrent is an emerging treatment method applying extremely low-level electric currents in the microampere range to treat various medical conditions, including Alzheimer's disease12, tension headaches13, sleep disorders14, depression15, congenital muscle torticollis16,17, musculoskeletal problems such as back pain18, knee pain19, and shoulder pain20 with wound healing21. The exact mechanisms of microcurrent therapy remain unknown, but it is thought to be associated with increased adenosine triphosphate (ATP) production, improved amino-acid transportation, and enhanced protein synthesis, thereby reducing inflammation and promoting tissue healing22,23. Several previous studies have revealed the effectiveness of microcurrent in managing hypertension by mitigating oxidative stress and exhibiting anti-inflammatory effects24,25. Furthermore, an animal study that investigated sinusitis in mice revealed that microcurrent treatment reduced inflammatory cytokine levels when compared to untreated sinusitis mice24. Therefore, microcurrent therapy can be a safe and non-invasive treatment option that could target hypertension and inflammation, thereby potentially offering an alternative approach to managing IADE and improving patient outcomes. However, as far as we have searched, there have been no pre-clinical studies that eval‎uated the efficacy and safety of microcurrent in treating IADE and related conditions. Additionally, no previous study has applied microcurrent to IADE.

This proof-concept study aims to assess the effect of microcurrent electrical stimulation on IADE development in the induced murine model. Each sham operation, induced dolichoectasia, and several treatment groups were compared.

두개내 동맥류(IADE)는

신장, 확장, 꼬임으로 특징지어지는 독특한 동맥병증입니다1,2,3.

IADE는

일반 인구에서 0.1%–6.5%의 유병률을 보였으며,

뇌졸중 환자의 경우 약 12%로 증가했습니다4.

IADE는

흔히 무증상일 수 있지만,

뇌경색, 뇌동맥류, 뇌출혈 또는 주변 구조물 압박을 포함한

심각한 뇌혈관 질환으로 나타날 수 있습니다5,6,7.

돌리코엑타시아증에 의한 허혈성 뇌졸중은

두 가지 주요 기전적 요인을 포함합니다:

관통하는 동맥의 기계적 신장과 차단,

그리고 동맥 확장과 관련된 혈류 감소로 인한 혈류 정체.

IADE의 병인은 연구되어 왔으며,

특히 고혈압과 관련된 중막에 영향을 미치는 동맥벽 손상의 일반적인 최종 경로로 설명될 수 있습니다6,8,9.

이전 연구1에서는

IADE의 발생이

동맥벽 내의 혈역학 스트레스 유발 염증 및 세포외기질(ECM) 리모델링과

밀접하게 관련되어 있다고 보고했습니다.

현재,

IADE는

주로 환자의 임상 증상 및 질병의 심각도에 따라 관리됩니다.

이 접근 방식에는

혈압 조절, 항혈전제 사용,

그리고 경우에 따라 혈관 내 시술 및 수술을 포함한 좀 더 침습적인 치료가 포함됩니다10,11.

항혈전제는

종종 이차성 허혈성 뇌졸중을 예방하기 위한 예방책으로 권장되지만,

돌리코엑타시아증 환자는 여전히 재발성 허혈성 뇌졸중의 위험이 증가합니다3.

돌기동맥류에 대한 외과적 개입은 접근하기가 어렵고,

시술 중 의인성 파열을 피하기 위해 집중적인 계획과 주의가 필요합니다11.

IADE에 대한 최적의 치료법에 대한 합의가 이루어지지 않았기 때문에,

근본적인 병태생리학적 요인을 고려한 안전하고 비침습적인 치료 옵션을 탐색해야 합니다.

이 연구는

미세전류 요법이

고혈압과 염증을 동시에 해결할 수 있기 때문에

IADE의 자연적인 경로를 수정할 수 있는 잠재적인 전략으로 간주했습니다.

미세 전류 요법은

극히 낮은 수준의 미세 전류를 사용하여

알츠하이머병12, 긴장성 두통13, 수면 장애14, 우울증15,

선천성 근육 토르티콜리16,17, 허리 통증18, 무릎 통증19, 어깨 통증20과 같은

다양한 의학적 상태를 치료하는 새로운 치료 방법입니다.

Microcurrent is an emerging treatment method applying extremely low-level electric currents in the microampere range to treat various medical conditions, including

Alzheimer's disease12,

tension headaches13,

sleep disorders14,

depression15,

congenital muscle torticollis16,17,

musculoskeletal problems such as back pain18, knee pain19, and shoulder pain20 with wound healing21

미세 전류 요법의 정확한 메커니즘은 아직 밝혀지지 않았지만,

아데노신 삼인산(ATP) 생성 증가,

아미노산 수송 개선,

단백질 합성 강화와 관련이 있는 것으로 여겨져,

염증을 줄이고 조직 치유를 촉진하는 것으로 알려져 있습니다22,23.

The exact mechanisms of microcurrent therapy remain unknown, but it is thought to be associated with increased

adenosine triphosphate (ATP) production,

improved amino-acid transportation, and

enhanced protein synthesis,

thereby reducing inflammation and promoting tissue healing

여러 연구에서 미세 전류가

산화 스트레스를 완화하고

항염 효과를 나타냄으로써

고혈압 관리에 효과가 있다는 사실이 밝혀졌습니다24,25.

또한, 쥐의 부비동염을 조사한 동물 연구에서

미세 전류 치료가 치료하지 않은 부비동염 쥐에 비해

염증성 사이토카인 수치를 감소시킨다는 사실이 밝혀졌습니다24.

따라서,

미세 전류 치료는

고혈압과 염증을 표적으로 삼을 수 있는

안전하고 비침습적인 치료 옵션이 될 수 있으며,

따라서

IADE 관리에 대한 대안적 접근 방식을 제공하고

환자 예후를 개선할 수 있는 잠재력을 가지고 있습니다.

그러나

지금까지 우리가 조사한 바에 따르면,

IADE 및 관련 질환의 치료에 미세 전류의 효능과 안전성을 평가한 전임상 연구는 없습니다.

또한,

IADE에 미세 전류를 적용한 연구는 없습니다.

이 개념 증명 연구는 유도된 쥐 모델에서

미세 전류 전기 자극이 IADE 발생에 미치는 영향을 평가하는 것을 목표로 합니다.

각 가짜 수술, 유도된 돌기형 확장증, 그리고 여러 치료 그룹을 비교했습니다.

Results

Microcurrent wave alleviates IADE-related arterial morphologic changes

Figure 1 shows the representative Hematoxylin and Eosin (H&E) images of arterial walls. The cerebral arterial lumen diameter was significantly higher in group 2-D (117.79 ± 17.05 µm) compared to group 1-C (76.64 ± 12.03 µm; p = 0.031) and group 3-M + D (77.29 ± 24.47 µm; p = 0.01). Cerebral arterial wall thickness in group 2-D (9.31 ± 2.26 µm) was significantly lower than in group 1-C (16.16 ± 1.6 µm; p = 0.012) and group 3-M + D (15.67 ± 2.86 µm; p = 0.004) (Fig. 2, Table 1). The arterial lumen diameter of group 4-D + M (100.28 ± 25.99 µm) was lower than that of group 2-D (117.79 ± 17.05 µm) (p = 0.641) and the arterial wall thickness of group 4-D + M (12.82 ± 5.17 µm) was higher than that of group 2-D (9.31 ± 2.26 µm) (p = 0.349) although with no significant difference.

Figure 1

Representative Hematoxylin and Eosin (H&E) staining and immunohistochemistry staining images of each group.

Full size image

Figure 2

Comparison of morphologic changes of the cerebral arterial wall measured by diameter, thickness, and extracellular matrix (ECM) composition. Comparison of diameter (A) and thickness (B) of cerebral artery. Comparison of ECM composition of the cerebral arterial wall, including smooth muscle cell (SMC) (C), elastin (D), and collagen (E). *p < 0.05, one-way ANOVA and post hoc Tukey test between the groups. ***p < 0.001, one-way ANOVA and post hoc Tukey test between the groups.

Full size image

Table 1 Comparison of the morphological changes in the cerebral artery among the five groups.

Full size table

Microcurrent wave preserves smooth muscle cells (SMC) and elastin while controlling collagen production in the IADE-induced arterial wall

Group 2-D showed a significantly lower proportion of SMC and elastin in the cerebral arterial wall (SMC: 38.05 ± 10.32% and elastin: 11.11 ± 6.97%) compared to groups 1-C (SMC: 70.93 ± 7.18% and elastin: 53.13 ± 9.08%), 3-M + D (SMC: 67.03 ± 6.17% and elastin: 47.22 ± 8.73%), and 4-D + M (SMC: 70.45 ± 9.35% and elastin: 51.2 ± 6.82%) (p < 0.001), respectively. Group 2-D (42.46 ± 14.12%) demonstrated a significantly higher proportion of collagen in the cerebral arterial wall compared to groups 1-C (6.94 ± 2.76%), 3-M + D (13.31 ± 4.67%), and 4-D + M (13.3 ± 3.84%) (p < 0.001), respectively (Fig. 2, Table 1). However, the proportion of SMC, elastin, and collagen in the cerebral arterial wall demonstrated no statistically significant differences among groups 1, 3, and 4 (Fig. 2, Table 1).

Cluster of differentiation 68 (CD68) staining and matrix metalloproteinase-8 (MMP-8) expression‎ by immunohistochemistry

Figure 3 shows representative immunohistochemically stained arterial walls. Immunohistochemical staining revealed distinctive high expression‎ patterns of CD68 and MMP-8 in group 2 compared to those in group 1. Furthermore, the expression‎ patterns of group 3 were somewhat morphologically similar to group 1, while that of group 4 was different from group 1. However, the expression‎ patterns of CD68 and MMP-8 demonstrated no significant difference, as examined by the scoring system among the groups.

Figure 3

Representative immunohistochemistry staining images of each group. Immunohistochemical staining revealed distinctive expression‎ patterns of CD68 and MMP-8 between groups 1 (grade 0/0) and 2 (grade 2/2). The expression‎ patterns of microcurrent treatment groups were variable: group 3 (CD68/MMP-8, grade 0/2), group 4 (grade 1/1), and group 5 (grade 1/1). The staining scoring system is defined as follows: immunohistochemistry grade 0: no stain, grade 1: less than half, grade 2: more than half of arterial wall circumference.

Full size image

Major organs of mouse do not show any sign of damage after microcurrent wave

Group 5-M revealed no signs of toxicity, according to histological observations in the liver and brain.

Discussion

This study revealed that microcurrent effectively prevents IADE progression, as evidenced by the improvement in morphology, such as the diameter and thickness of the cerebral artery, and the prevention of ECM degeneration. To the best of our knowledge, this study is the first to reveal the effect of microcurrent on IADE development. Dolichoectatic arteries are characterized by a decreased arterial wall thickness and increased arterial lumen diameter, resulting in tortuous, elongated, and widened cerebral arterial configuration5. Previous studies that used the elastase-induced IADE mouse model revealed that arterial lumen dilatation occurs due to internal elastic lamina loss, concerning the inflammatory process and increased MMP expression‎. These MMPs, released by activated macrophages, digest elastin, leading to SMC apoptosis and the artery’s mechanical weakening1,26,27. The present study revealed that the IADE group showed dilated cerebral arteries lumen diameter and thinned cerebral arteries wall compared to the control group, which is in concordance with previous results.. Conversely, the preoperatively applied microcurrent significantly restored the morphology of the cerebral arteries compared to the IADE group. Therefore, the microcurrent can be recognized to affect abnormal cerebral artery deformation in IADE progression and homeostatically restore the abnormal arteries. These results indicate that microcurrent could be a potential therapeutic approach for managing IADE and its related cerebral artery abnormalities.

Microcurrent therapy has treated various diseases, such as musculoskeletal conditions, wounds, and pain. One of its advantages is that it provides subsensory stimulation, indicating that it is < 1 mA, which results in no discomfort for patients compared to other conventional electrical stimulations such as transcutaneous electrical nerve stimulation.18 Moreover, many other clinical and animal studies reported no severe adverse effects of microcurrent16,18,19,23,28,29. The exact mechanism of microcurrent therapy remains unclear, but it may be related to increasing the adenosine triphosphate generation, facilitating amino-acid transportation, and promoting protein synthesis to reduce inflammation and induce tissue healing.22 Additionally, microcurrent may regulate the disturbed homeostasis of intracellular Ca2+ in injured tissues, thereby reducing muscle shortening and improving maximum force production in muscles with delayed-onset muscle soreness30. McMakin et al.31 revealed that a substantial reduction of inflammatory markers, including interleukin-1 (IL-1), IL-6, tumor necrosis factor-alpha (TNF-α), and the neuropeptide substance P was observed in patients with fibromyalgia treated with microcurrent. Microcurrent therapy has been increasingly utilized for various medical conditions due to its effectiveness and non-invasiveness, along with minimal side effects and discomfort. Thus, we selected microcurrent as the treatment for IADE in our study.

Hypertension or hemodynamic stress also contributed to inducing endothelial cell dysfunction, inflammatory cell infiltrations, SMC breakdown, and ECM remodeling, ultimately leading to vessel wall degeneration and cell death32,33. Hence, the management of arterial hypertension is crucial in dealing with dolichoectasia. Lin et al.34 reported hypertension as a major contributing factor in the course of IADE formation and caused the increased incidence of ischemic stroke and cerebral hemorrhage. Cerebral infarction in IADE cases is caused by luminal thrombi blocking small branches of the arteries. Furthermore, the thin and dilated arterial wall may be broken and bulged with a developing aneurysm, which poses a risk for intracranial hemorrhage35. Conventional treatment for ischemic stroke involves long-term anti-platelet agents, anti-coagulation agents, or invasive interventions. However, these thrombolytic or anticoagulant therapies could be a risk factor for aneurysm rupture and intracranial hemorrhage. The advent of the new treatment concept has been anticipated because the control of hypertension becomes crucial in managing IADE which carries higher risks of ischemic stroke, cerebral aneurysm, or intracranial hemorrhage9,34.

Microcurrent may be a beneficial treatment option for hypertension by managing oxidative stress, which is a major factor in cardiovascular disease development. Zalba et al.36 indicated that various factors, such as humoral, genetic, and hemodynamic elements, activate NAD(P)H oxidase, thereby increasing the production of superoxide anion, which is associated with endothelial dysfunction and media hypertrophy. Reactive oxygen species (ROS), including superoxide anion, play a crucial role in intracellular signaling and may contribute to vascular SMC (VSMC) hypertrophy and hyperplasia. Thus, high blood pressure regulation by reducing ROS appears promising. Lee et al.25 revealed that microcurrent application could stabilize mitochondria, act as antioxidants, and enhance vascular tissue function, thereby potentially aiding hypertension control. A previously unpublished study revealed that microcurrent effectively lowered blood pressure by reducing liver fat metabolism caused by hyperglycemia and renin levels. The present study revealed that the preoperative application of microcurrent resulted in significantly shorter cerebral arterial diameters and thicker arterial walls compared to the IADE group. The IADE group and the group with microcurrent applied after inducing IADE demonstrated no significant difference, but the latter group exhibited a trend of lower diameter and higher thickness. Blood pressure and ROS were not assessed in this study, but these results indirectly indicate that microcurrent may affect IADE by influencing hypertension. Further research is needed to understand the exact evidence and mechanism of how microcurrent controls hypertension and its potential effects on IADE.

The results of our study revealed that SMC, elastin, and collagen components in the groups where microcurrent was applied pre- or postoperatively were maintained at similar levels to the healthy control group and significantly different compared to the IADE group. This observation is congruent with a previous study reporting that microcurrent restores the normal function of vascular tissues25. The arterial walls consist of ECM components, including SMC, elastin, and collagen, with elastin being a key factor in IADE formation. The single elastase injection in the cerebrospinal fluid (CSF) space reduced elastin and broke down the normal arterial wall composition. Our previous study1 revealed a lower proportion of elastin and higher proportions of collagen and SMC in the dolichoectactic artery. Liu et al.37 revealed elastin degradation and increased type 1 collagen, thereby contributing to stiffness of the arterial wall in coronary arteria ectasia. Conversely, Kapeller et al.38 revealed that microcurrent affects an alteration in the extracellular matrix and reduces collagen type 1 in myocardial specimens of spontaneously hypertensive rats. Additionally, microcurrent supported early wound healing by stimulating the regeneration of vessels affected by cigarette smoke, which is also a risk factor for IADE39. These findings indicate that the microcurrent may help maintain and normalize arterial vasculature, thereby contributing to IADE prevention and management.

Inflammation is a significant factor that influences arterial wall dilatation, and numerous studies have implicated macrophages and monocytes as important agents in inflammation and IADE formation40,41,42. Our study revealed that the IADE groups demonstrated a significantly lower elastin concentration and a higher proportion of collagen in the arterial wall. Upregulated secretion of MMP within the arterial wall seems to mediate elastic fiber degradation and reduction in elastin proportions. As elastin was progressively lost, the collagens remained highly concentrated, contributing to dolichoectasia40. Furthermore, Laxton et al.43 revealed low angiotensin I and high angiotensin II in MMP-8 abundant mice, probably indicating that MMP-8 is involved in the conversion process of angiotensin I to II. Additionally, MMP-8 functions as a generator for angiotensin II production, leading to increased blood pressure. MMP-8 is also involved in vascular inflammation through angiotensin II44. Therefore, the curtailment of MMP might be effective in treating IADE, considering the pathophysiology. CD68, a marker of macrophages, is also related to inflammation. Other previous studies revealed some association of CD68 with cerebral aneurysm formation and rupture45,46. Our previous study revealed a significant increase in CD68 in the IADE model as the inflammation occurred in the IADE formation, and treatment with melittin-loaded L-arginine-coated iron oxide nanoparticles reduced inflammation and prevented IADE formation. Our present study revealed no significant difference in the scoring system of the expression‎ patterns of MMP-8 and CD68 between the IADE group and the microcurrent applied groups, but we observed gross morphological differences between the disease and treatment groups. The small sample size may have caused the lack of significant differences in the scoring system. Further studies with larger sample sizes are required to fully eval‎uate the quantitative measurements of inflammation and the mechanism of microcurrent in reducing inflammation.

The present study has several limitations. First, the sample size was relatively small, which may have affected the statistical significance of the results. Additionally, brain tissue from mice is quite small, and technical errors during tissue cutting or trimming could cause the loss of the cerebral arteries in the specimens, thereby potentially affecting the analysis. Second, we did not perform diverse frequencies and durations of microcurrent. We selected the present microcurrent type referred to in the previous study47, but further investigations with diverse microcurrents are needed to determine the most effective type of microcurrent therapy for IADE. Third, this study included no clinical assessment. Future studies should consider additional parameters, such as neurologic signs or symptoms for clinical application. Fourth, we did not include a group that continuously applied microcurrent from preoperation to postoperation. Such a group could provide valuable insights into the impact of all areas from prevention to microcurrent treatment. Fifth, we did not contemplate endothelial cell dysfunction and chronic inflammation markers such as hsCRP and fibrinogen. Sixth, while we noted enhancements in the tunica intima and tunica media following microcurrent therapy, our study's scope is limited by the lack of biomarker data for the tunica intima, warranting further investigation, Seventh, we relied solely on immunohistochemistry (IHC) for inflammation eval‎uation. Future studies might consider additional methods, such as Western blot and polymerase chain reaction, for a more quantitative eval‎uation of inflammation. Finally, given the safety profile and efficacy of this microcurrent therapy, Microcurrent may be a promising preemptive strategy to protect against IADE development. Further clinical feasibility studies will be required in high-risk populations for IADE development.

토론

이 연구는

미세 전류가 IADE의 진행을 효과적으로 방지한다는 것을 밝혀냈습니다.

이는 대뇌동맥의 직경과 두께와 같은 형태학적 개선과 ECM 퇴화 방지를 통해 입증되었습니다.

저희가 아는 한,

이 연구는 미세 전류가 IADE의 발달에 미치는 영향을 밝혀낸 최초의 연구입니다.

장경동맥은

동맥벽 두께가 감소하고 동맥 내강 직경이 증가하여

구불구불하고 길쭉하며 넓어진 대뇌동맥의 형태를 띠는 것이 특징입니다5.

엘라스타제 유발 IADE 마우스 모델을 사용한 이전 연구에서는 염증 과정과 MMP 발현 증가와 관련하여 내부 탄성층 손실로 인해 동맥 내강이 확장되는 것으로 나타났습니다.

활성화된 대식세포에 의해 방출된 이러한 MMP는 엘라스틴을 분해하여 SMC의 세포 사멸과 동맥의 기계적 약화를 유발합니다1,26,27.

본 연구는 IADE 그룹이 대조군에 비해 확장된 대뇌동맥 내강 직경과 얇아진 대뇌동맥 벽을 보였으며, 이는 이전의 결과와 일치하는 것으로 나타났습니다. 반대로, 수술 전 적용된 미세 전류는 IADE 그룹에 비해 대뇌동맥의 형태를 현저하게 회복시켰습니다. 따라서 미세 전류는 IADE 진행 과정에서 비정상적인 대뇌동맥 변형에 영향을 미치고 비정상적인 동맥을 항상성적으로 회복시키는 것으로 인식될 수 있습니다. 이러한 결과는 미세 전류가 IADE 및 관련 대뇌동맥 이상을 관리하는 잠재적인 치료 접근법이 될 수 있음을 나타냅니다.

미세 전류 요법은

근골격계 질환, 상처, 통증 등

다양한 질병을 치료하는 데 사용되어 왔습니다.

이 요법의 장점 중 하나는

감각을 거의 느끼지 못하는 자극을 제공한다는 점인데,

이는 1mA 미만으로,

경피적 전기 신경 자극과 같은 다른 기존의 전기 자극에 비해

환자에게 불편함을 유발하지 않는다는 것을 의미합니다.18

또한, 미세 전류 요법의 심각한 부작용은 없다고 보고된 많은 다른 임상 및 동물 연구가 있습니다16,18,19,23,28,29.

미세 전류 요법의 정확한 메커니즘은 아직 명확하지 않지만,

아데노신 삼인산 생성을 증가시키고,

아미노산 수송을 촉진하며,

단백질 합성을 촉진하여

염증을 줄이고 조직 치유를 유도하는 것과 관련이 있을 수 있습니다22.

또한,

미세 전류는

손상된 조직에서 세포 내 Ca2+의 교란된 항상성을 조절하여

근육 수축을 줄이고

지연 발병성 근육통이 있는 근육의 최대 힘 생성을 향상시킬 수 있습니다30.

McMakin et al.31은

미세 전류 요법으로 치료받은 섬유근육통 환자들에서

인터루킨-1(IL-1), IL-6, 종양 괴사 인자-알파(TNF-α), 신경펩티드 물질 P를 포함한

염증성 마커의 상당한 감소가 관찰되었다고 밝혔다.

미세 전류 요법은 그 효과와 비침습성, 그리고 부작용과 불편함이 적다는 장점 때문에 다양한 의학적 조건에 점점 더 많이 활용되고 있다. 따라서, 우리는 IADE 치료법으로 미세 전류를 선택했습니다.

고혈압이나 혈역학적인 스트레스도 내피세포 기능 장애, 염증세포 침윤, SMC 파괴, 그리고 ECM 리모델링을 유발하는 데 기여하여, 궁극적으로 혈관벽 퇴화 및 세포 사멸을 초래합니다32,33.

따라서, 동맥고혈압의 관리는 동맥확장증 치료에 매우 중요합니다. Lin et al.34은 고혈압이 IADE 형성 과정에서 주요한 기여 요인으로 작용하며, 허혈성 뇌졸중과 뇌출혈의 발생률을 증가시킨다고 보고했습니다. IADE 사례에서 뇌경색은 동맥의 작은 가지들을 막는 관상 혈전에 의해 발생합니다. 또한, 얇고 확장된 동맥벽이 파열되어 동맥류가 발생할 수 있으며, 이 경우 두개내 출혈의 위험이 있습니다35. 기존의 허혈성 뇌졸중 치료는 장기적인 항혈소판제, 항응고제, 또는 침습적 개입을 포함합니다. 그러나 이러한 혈전 용해제 또는 항응고제 치료는 동맥류 파열과 두개내 출혈의 위험 요소가 될 수 있습니다. 허혈성 뇌졸중, 뇌동맥류, 또는 두개내 출혈의 위험이 더 높은 IADE를 관리하는 데 고혈압의 조절이 중요해지기 때문에 새로운 치료 개념의 출현이 기대되고 있습니다9,34.

미세 전류는 심혈관 질환 발병의 주요 원인인 산화 스트레스를 관리함으로써 고혈압에 유익한 치료 옵션이 될 수 있습니다. Zalba et al.36은 체액성, 유전적, 혈역학적 요소와 같은 다양한 요인이 NAD(P)H 산화효소를 활성화시켜 내피 기능 장애 및 혈관 비대와 관련된 슈퍼옥사이드 음이온의 생성을 증가시킨다고 지적했습니다. 슈퍼옥사이드 음이온을 포함한 활성산소(ROS)는 세포 내 신호 전달에 중요한 역할을 하며, 혈관 내피세포(VSMC)의 비대 및 증식에 기여할 수 있습니다. 따라서, 활성산소를 줄임으로써 고혈압을 조절하는 것은 유망한 것으로 보입니다. Lee et al.25은 미세 전류를 적용하면 미토콘드리아를 안정화하고 항산화 작용을 하며 혈관 조직 기능을 향상시킬 수 있으므로 고혈압 조절에 도움이 될 수 있다고 밝혔습니다. 미세 전류를 이용한 치료가 고혈당과 레닌 수치로 인한 간 지방 대사를 감소시켜 혈압을 효과적으로 낮춘다는 사실이 이전에 발표되지 않은 연구에서 밝혀졌습니다. 이 연구에서는 미세 전류를 수술 전에 적용한 경우, IADE 그룹에 비해 뇌동맥 직경이 현저히 짧아지고 동맥벽이 두꺼워지는 결과가 나타났습니다. IADE 그룹과 IADE 유도 후 미세 전류를 적용한 그룹 간에는 유의미한 차이가 나타나지 않았지만, 후자의 경우 직경이 더 작고 두께가 더 두꺼운 경향을 보였습니다. 이 연구에서는 혈압과 ROS를 평가하지 않았지만, 이 결과는 미세 전류가 고혈압에 영향을 미침으로써 IADE에 영향을 미칠 수 있음을 간접적으로 보여줍니다. 미세 전류가 고혈압을 조절하는 방법과 IADE에 미치는 잠재적 영향에 대한 정확한 증거와 메커니즘을 이해하기 위해서는 추가 연구가 필요합니다.

이 연구의 결과에 따르면, 수술 전후에 미세 전류를 적용한 그룹의 SMC, 엘라스틴, 콜라겐 성분은 건강한 대조군과 비슷한 수준으로 유지되었고, IADE 그룹과 비교했을 때 현저하게 다른 것으로 나타났습니다. 이 관찰 결과는 미세 전류가 혈관 조직의 정상적인 기능을 회복시킨다는 이전 연구 결과와 일치합니다25. 동맥벽은 SMC, 엘라스틴, 콜라겐을 포함한 ECM 성분으로 구성되어 있으며, 엘라스틴은 IADE 형성의 핵심 요소입니다. 뇌척수액(CSF) 공간에 단일 엘라스타제 주사를 주입하면 엘라스틴이 감소하고 정상적인 동맥벽 구성이 파괴됩니다. 이전 연구1에서는 돌리코엑타크틱 동맥에서 엘라스틴의 비율이 낮고 콜라겐과 SMC의 비율이 높은 것으로 나타났습니다. Liu et al.37은 엘라스틴의 분해와 1형 콜라겐의 증가를 밝혀내어, 관상동맥 확장증의 동맥벽 경직에 기여하는 것으로 나타났습니다. 반대로, Kapeller et al.38은 미세 전류가 자발성 고혈압 쥐의 심근 표본에서 세포 외 기질의 변화에 영향을 미치고 1형 콜라겐을 감소시킨다는 것을 밝혀냈습니다. 또한, 미세 전류는 담배 연기에 의해 영향을 받은 혈관의 재생을 자극함으로써 조기 상처 치유를 지원했는데, 이는 IADE의 위험 요소이기도 합니다39. 이러한 연구 결과는 미세 전류가 동맥 혈관 구조를 유지하고 정상화함으로써 IADE 예방과 관리에 기여할 수 있음을 보여줍니다.

염증은 동맥벽 확장에 영향을 미치는 중요한 요소이며, 수많은 연구에서 대식세포와 단핵구가 염증과 IADE 형성에 중요한 역할을 한다는 사실이 밝혀졌습니다40,41,42. 우리의 연구에 따르면 IADE 그룹은 동맥벽에서 엘라스틴 농도가 현저히 낮고 콜라겐 비율이 더 높은 것으로 나타났습니다. 동맥벽 내 MMP의 분비 증가가 탄성섬유 분해와 엘라스틴 비율 감소를 매개하는 것으로 보입니다. 엘라스틴이 점진적으로 소실됨에 따라 콜라겐은 고농도로 유지되어, 돌기형 확장증에 기여합니다40. 또한, Laxton 등43은 MMP-8이 풍부한 마우스에서 낮은 안지오텐신 I과 높은 안지오텐신 II를 발견했는데, 이는 아마도 MMP-8이 안지오텐신 I이 안지오텐신 II로 전환되는 과정에 관여한다는 것을 나타내는 것으로 보입니다. 또한, MMP-8은 안지오텐신 II 생성을 촉진하는 기능을 수행하여 혈압을 상승시킵니다. MMP-8은 또한 안지오텐신 II를 통해 혈관 염증에 관여합니다44. 따라서, 병리 생리학을 고려할 때, MMP의 억제는 IADE 치료에 효과적일 수 있습니다. 대식세포의 표지자인 CD68도 염증과 관련이 있습니다. 다른 이전 연구에서도 CD68과 뇌동맥류 형성 및 파열의 연관성이 밝혀졌습니다45,46. 우리의 이전 연구에서는 염증이 IADE 형성에서 발생함에 따라 IADE 모델에서 CD68이 유의하게 증가하는 것으로 나타났으며, 멜리틴이 함유된 L-아르기닌 코팅 산화철 나노입자를 사용한 치료는 염증을 감소시키고 IADE 형성을 예방했습니다. 현재의 연구에서는 IADE 그룹과 미세 전류를 적용한 그룹 사이에 MMP-8과 CD68의 발현 패턴 점수 체계에 유의미한 차이가 없었지만, 질병과 치료 그룹 사이에 큰 형태학적 차이를 관찰했습니다. 표본의 크기가 작기 때문에 점수 체계에 유의미한 차이가 없었을 수 있습니다. 염증의 정량적 측정과 염증을 줄이는 미세 전류의 메커니즘을 완전히 평가하기 위해서는 표본의 크기가 더 큰 추가 연구가 필요합니다.

이번 연구는 몇 가지 한계점이 있습니다. 첫째, 표본 크기가 상대적으로 작아서 결과의 통계적 유의성에 영향을 미쳤을 수 있습니다. 또한, 생쥐의 뇌 조직은 매우 작고, 조직을 자르거나 다듬는 과정에서 기술적 오류가 발생하면 표본에서 대뇌동맥이 손실되어 분석 결과에 영향을 미칠 수 있습니다. 둘째, 다양한 주파수와 지속 시간의 미세 전류를 사용하지 않았습니다. 이전 연구47에서 언급된 현재의 미세 전류 유형을 선택했지만, IADE에 가장 효과적인 미세 전류 요법 유형을 결정하기 위해서는 다양한 미세 전류에 대한 추가 연구가 필요합니다. 셋째, 이 연구에는 임상 평가가 포함되지 않았습니다. 향후 연구에서는 임상 적용을 위한 신경학적 징후 또는 증상과 같은 추가 매개 변수를 고려해야 합니다. 넷째, 수술 전부터 수술 후까지 지속적으로 미세 전류를 적용한 그룹을 포함시키지 않았습니다. 이러한 그룹은 예방에서 미세 전류 치료에 이르기까지 모든 영역에 미치는 영향에 대한 귀중한 통찰력을 제공할 수 있습니다. 다섯째, 우리는 내피세포 기능 장애와 hsCRP 및 피브리노겐과 같은 만성 염증 마커를 고려하지 않았습니다. 여섯째, 미세 전류 요법 후 내피층과 중간층이 개선된 것을 확인했지만, 본 연구의 범위는 내피층에 대한 바이오마커 데이터가 부족하여 제한적이며, 추가 연구가 필요합니다. 일곱째, 우리는 염증 평가를 위해 면역조직화학(IHC)에만 의존했습니다. 향후 연구에서는 염증의 정량적 평가를 위해 웨스턴 블롯과 중합효소 연쇄 반응과 같은 추가적인 방법을 고려할 수 있습니다. 마지막으로, 이 미세 전류 요법의 안전성 프로필과 효능을 고려할 때, 미세 전류 요법은 IADE 발생을 예방하는 유망한 선제적 전략이 될 수 있습니다. IADE 발생 위험이 높은 집단을 대상으로 추가적인 임상 타당성 연구가 필요합니다.

Conclusion

This pilot study revealed the effectiveness of microcurrent therapy in preventing and positively affecting IADE development by improving the morphology and composition of cerebral arteries compared to the non-treatment group. These results indicate that microcurrent could be a promising and non-invasive treatment option for IADE. Further comprehensive studies are necessary to fully comprehend the mechanism underlying microcurrent therapy and its potential clinical application. Overall, this study establishes a solid groundwork for future research and clinical investigations of microcurrent therapy as a potential IADE treatment.

결론
이 파일럿 연구는 미세 전류 요법이 치료하지 않은 그룹과 비교하여 대뇌 동맥의 형태와 구성을 개선함으로써 IADE의 발생을 예방하고 긍정적인 영향을 미치는 효과가 있음을 보여주었습니다. 이러한 결과는 미세 전류가 IADE에 대한 유망한 비침습적 치료 옵션이 될 수 있음을 보여줍니다. 미세 전류 요법의 기전과 잠재적인 임상 적용을 완전히 이해하기 위해서는 더 포괄적인 연구가 필요합니다. 전반적으로, 이 연구는 미세 전류 요법이 잠재적인 IADE 치료법으로 사용될 수 있다는 가능성을 뒷받침하는 견고한 기초를 마련해 줍니다.

Methods

Experimental reagents

We purchased angiotensin II (A9525) and elastase (E7885) from Sigma-Aldrich (St. Louis, Missouri, USA) and Alzet osmotic pumps (200 µL, 0.5 µL/h) from Durect corporation (Cupertino, California, USA). The 10 µL model 701 syringe with a 26-G 2.0-inch point-style-3 Hamilton replacement needle was supplied by Fisher Scientific (Hampton, New Hampshire, USA). We purchased IHC antibodies from Abcam (Cambridge, UK) and Cell Signaling Technology (Danvers, Massachusetts, USA).

Animal grouping and IADE model generation

This animal study was approved by the Institutional Animal Care and Use Committee (IACUC) of the Catholic University of Daegu School of Medicine (Approved number: DCIAFCR-221027-28-YR)), which is the author’s affiliation, in compliance with IACUC guidelines for the care and use of animals. In addition, this study was conducted in accordance with ARRIVE guidelines. This study randomly allocated 20 mice using computerized random numbers into five groups: group 1-C (healthy control), group 2-D (IADE model), Group 3-M + D received microcurrent therapy for a total of 2 weeks, beginning one week before nephrectomy and continuing through to brain surgery to prevent IADE, group 4-D + M (microcurrent administration for 4 weeks after brain surgery for IADE treatment), and group 5-M (microcurrent administration for 4 weeks to eval‎uate toxicity) (Fig. 4).

Figure 4

Timeline of the study. Twenty mice were randomly allocated into 5 groups: group 1 (healthy control); group 2 (the intracranial arterial dolichoectasia [IADE] model); group 3 (microcurrent therapy for a total of 2 weeks, beginning one week before nephrectomy and continuing through to brain surgery to prevent IADE); group 4 (microcurrent therapy administered for 4 weeks after the brain surgery for treatment of IADE); group 5 (microcurrent therapy administered for 4 weeks from 0th week for toxicity eval‎uation, when the brain surgery was performed in the other group).

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The 6-week-old C57/BL6 mice (Hyo-Chang Science, Korea) were used to create the IADE model. The breeding room maintained a 12-h light–dark cycle to artificially create day and night environments, with free access to normal feeding and water supply. The mice were allowed to adapt to the new environment for 1 week preoperatively. One week before the brain surgery, a unilateral nephrectomy was performed to induce hypertension or hemodynamic stress, following the procedures as outlined below. Preoperatively, the mice were anesthetized with isoflurane inhalation anesthesia. The skin on the unilateral posterolateral side of the back was shaved. An incision was made perpendicular to the spine after finding the spleen to determine the location of the kidney. The skin was then removed, and an incision was made in the abdominal wall. Light pressure was applied on either side of the incision to expose the kidney within the body wall. The left kidney was surgically removed, the abdominal wall was then sutured, and the skin wound was closed with clips (Fig. 5-A)..

Figure 5

Schematic diagram of IADE model generation. A. Hypertension or hemodynamic stress was induced by unilateral nephrectomy 1 week before brain surgery. B. Elastase was injected stereotaxically into the right basal cistern and angiotensin II was continuously infused by subcutaneous osmotic pump implantation at 0 week. C. Microcurrent therapy was performed.

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The brain surgery was conducted 1 week after the nephrectomy as follows: a single dose of elastase (4 μL of 2.5 mU/μL) solution was injected stereotaxically into the ventricular zone of the mice at 0.2 μL/min. The coordinates of the ventricular zone were determined based on the mouse brain’s Atlas. The bregma was determined by the coronal suture and sagittal suture intersection. The syringe was manipulated 0.2 mm posteriorly from the bregma, 1.0 mm laterally to the right, and 2.4 mm ventrally from the skull surface. After the injection, an angiotensin II pump was subcutaneously implanted at the back of the mouse to create the IADE model (Fig. 5B).

The mice were subjected to cardiac perfusion with 4 mL of phosphate-buffered saline (PBS), 4 mL of 4% paraformaldehyde (PFA), and 4 mL of bromophenol blue dye solution dissolved in 10% (w/v) gelatine/PBS, at week 4 after microcurrent treatment. The brains were carefully isolated from the skull using forceps. The brain tissues were immersed in 4% PFA in the refrigerator (4 °C) for at least one day before conducting histopathologic analysis.

Microcurrent therapy

Microcurrent was applied for 12 h a day on weekdays according to the specific period set for each group to investigate its therapeutic effect (Figs. 4, 5C). A connection wire from the microcurrent generator (Natural Well Tech, Busan, Korea) was used to deliver the current through a copper plate of the same size as the cage floor. This setup enabled the mice to receive the current through their feet touching the floor, thereby allowing the current to reach the brain (Fig. 6). Microcurrent is usually applied during the active time of humans, which is daytime, thus microcurrent was applied during the mice’s active time, which is the nighttime cycle for nocturnal animals. There are various waveforms of microcurrent, but Kim et al.47 revealed that all waveforms had an effect, and the step form waveform showed significant effects on both clinical parameters, such as cognition and protein production related to Alzheimer’s disease in mice models. Therefore, we selected the microcurrent with the step form waveform (0, 1.5, 3, and 5 V) with wave superposition. The intensity of the microcurrent was set to 1 μ A (250 Ω), the voltage was set to 5 V, and the basic frequency was set to 7 Hz with an additional 44 KHZ frequency superimposition.

Figure 6

Microcurrent therapy. A connection wire from the microcurrent generator was used to deliver the current through a copper plate of the same size as the cage floor. This setup enabled the mice to receive the current through their feet touching the floor, allowing the current to reach the brain.

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Histopathologic analysis

All the histological parameters of the study were measured by an examiner (M.H.P.) who was unaware of the group allocation. Specimens were paraffin-embedded after at least one day of PFA fixation. The paraffin blocks were then sliced into five sections based on the location of the circle of Willis of cerebral arteries. H&E staining was performed to measure the vessel thickness and diameter for morphological examination. IHC was also conducted to analyze elastin, collagen, and SMCs using corresponding staining, such as Verhoeff Van Gieson, Masson’s trichrome, and alpha-smooth muscle actin (α-SMA), respectively. Furthermore, IHC of CD68, TNF-α, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and MMP8 were conducted to examine inflammatory responses in the cerebral arteries. After mounting the tissue on the side, they were scanned by a Panoramic slide scanner to magnify H&E 200 × and IHC 400 × .

A scoring system was introduced to categorize the expression‎ levels of pro-inflammatory mediators (CD68, TNF-α, NF-κB, and MMP-8) in the cerebral arterial wall. Grade 0 denoted the absence of marker expression‎ in the cerebral artery, grade 1 indicated a marker expression‎ below the halfway point of the arterial wall circumference, and grade 2 represented a marker expression‎ above the halfway point, encompassing more than half of the arterial wall circumference.

Measurement of diameter and thickness

A ruler within the Panoramic Slide Scanner viewer program was used to measure the arterial diameter. The length of the cerebral arterial diameter was calculated by taking the average value between two horizontal points and two vertical points along the vessel. The cerebral arterial thickness was determined by dividing each vessel into four equal parts. The average value of the representative thickness in each part was calculated, providing an overall measurement of the vessel’s thickness (Fig. 7).

Figure 7

Measurements of cerebral artery lumen diameter and wall thickness. The length of the lumen diameter was calculated as the average value of the representative length of two horizontal points and two vertical points of the vessel, and the wall thickness was calculated as the average value of the representative thickness of each part of the vessel equally divided into four parts.

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Analysis of extracellular matrix components

The analysis was performed following the method presented in the previous study1. After removing the background using the Adobe Photoshop program, the extracellular matrix components of the cerebral arterial wall, such as SMCs, elastin, and collagen, were assessed by measuring the selected ROI within the image using ImageJ software. The area of the ROI was measured and then divided by the area of the entire cerebral artery to quantify the components. This calculation enabled the estimation of the percentage of each component present in the cerebral artery.

Statistical analysis

Statistical analyses were conducted using the IBM Statistical Package for the Social Sciences program for Windows version 22.0 (IBM Corp., Armonk, New York, USA). For calculating sample size, we conducted pilot study. The primary end point is the cerebral arterial diameter. In pilot study, we used one mouse in each group, therefore four randomly selected field was eval‎uated in each group. The effect size was 0.49, and for such effect size, to achieve a power of at least 95% using the ANOVA with a significance level of 0.05, at least 76 fields were needed. Four fields can be obtained from one mouse. Therefore, 16 mouse were needed. Considering drop rate as 20%, we determined sample size as 20. One-way analysis of variance (ANOVA) was used to determine intra- and inter-group statistical differences. A post hoc Tukey test was additionally performed when one-way ANOVA demonstrated significant differences between the groups. The mean values were followed by 95% confidence intervals, and all the data were expressed as the means ± standard deviations. The statistically significant levels were pre-determined at P-values of < 0.05. The post-hoc power analysis was performed, and the power was calculated as > 0.95.

Ethical statement

The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted according to the guidelines of the IACUC, and approved by the Catholic University of Daegu School of Medicine Animal Care and Use Committee.

Values are presented as mean ± standard deviation.

SMC: smooth muscle cell.

*p < 0.05, one-way ANOVA test among groups.

†p < 0.05, post hoc Tukey test between groups 1 and 2.

‡p < 0.05, post hoc Tukey test between groups 2 and 3.

♦p < 0.05, post hoc Tukey test between groups 2 and 4.

Data availability

All data generated or analyzed during this study are included in this published article.

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