1. Introduction

Globally, stroke is the second leading cause of death above the age of 60 years, and the fifth leading cause of death in people aged 15 to 59 years old. There were 15 million people worldwide suffer of new stroke events that caused nearly six million of deaths and another five million are left permanently disabled. ^[1] The incidence of ischemic stroke was more than 80% with the rest was hemorrhagic stroke. ^[2] In Indonesia, stroke was ranked as the first leading cause of death.^[3] Stroke was not only attacking the elderly population but also young and productive ones, causing physical and psychological burden for patients, families and communities. Disability from a stroke would have an impact on declining productivity and economic capacity of community as well as the nation. ^[4]

The mechanism of brain injury in ischemic stroke was a very complex process and associated with an inflammatory process involving neuronal cells, glial cells, endothelial cells, extracellular matrix, and peripheral leukocytes. ^[5] Activated astrocytes were able to produce anti-inflammatory cytokines such as Transforming Growth Factor Beta (TGFβ). TGFβ was a major regulatory molecule for immune response, which may lower the immune response after injury and regulate immune cell phenotype to reduce the inflammatory phase. TGFβ was also a growth factor that affects growth, homeostasis, angiogenesis and tissue repair. ^{[6, 7]} Intranasal delivery of TGF-β1 in mice after stroke reduces infarct volume and increases neurogenesis in the subventricular zone. ^[8] According to Dobolyi et al, TGFβ has five dominant neuroprotective effects such as anti-inflammatory, anti-apoptotic, protection against excitotoxicity, triggering angiogenesis and neuronal regeneration. ^[9]

TGFβ was 25000 dalton’s homodimeric protein, which has 3 types of isoforms namely TGFβ1, TGFβ2, and TGFβ3. TGFβ1 was a specific isoform induced by injury. TGFβ was produced and secreted by transformed blood cells such as lymphocytes, monocytes, and platelets. In the brain, all main cells have a TGFβ receptor, but TGFβ signaling mainly increased in post-ischemic astrocytes and microglia. ^{[10, 11, 12]}

In this study, the author intends to explore the role of TGFβ-1 in acute ischemic stroke pathomechanism, and its role in predicting clinical outcomes.

2. Research Methods

The research was conducted during the period of August 2014 - January 2015 using a longitudinal cohort design.The rated of clinical outcome was assessed after 1 month post-stroke. The study population was all patients with anterior system ischemic stroke who comes within 72 hours after onset and treated at Siloam Hospitals Lippo Village. Patients with a history of previous stroke, thrombolysis therapy, impaired liver function, impaired renal function, Congestive Heart Failure, Acute Myocardial Infarct, or pneumonia were excluded from the study. The diagnosis of anterior system acute ischemic stroke was done by using head DW-MRI. Venous blood sampling was taken from subjects who met the inclusion criteria to measure levels of TGF-β1 serum using Human TGF-β1 Immunoassay Quantikine® (R & D Systems, Inc., USA). Blood sampling performed at ≤ 72 hours of onset and 7^th days post stroke and the clinical degree was assessed at ≤ 72 hours of onset, 7^th days and 30^th days post stroke using the NIH Stroke Score. This study was granted an etichal approval from Mochtar Riyadi Institute of Nanotechnology Ethic Committee No. 082 / MRIN-EC / 09/2014. Informed consents from all subjects were collected prior to data collection.

3. Result

Sixty four subjects were obtained the inclusion criteria. There were 46 men (62%) and 18 women (29%). Among these subjects, 16 persons were examined twice for their levels of TGF-β1; at 72 hours and 7^th days of stroke onset. The samples’ age was ranged from 37 to 89 years old with mean age was58.78 ± 11.31. The majority age group was 50-59 years old(46.8%). Most of subjects (93.75%) had hypertension. Clinical classification of LACI (lacunar infarct anterior circulation) and moderate initial clinical degree (NIHSS score 4-15) with an average score of 5.2 ± 3.74 was found in 76.56% and 59.4% of all subjects respectively. Table 1 and Table 2 shows the general characteristics of subjects in this study.

Table 1. Characteristic of Subjects

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Table 2. Characteristic of Subjects

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The clinical degree of acute stroke patients was measured using the NIH Stroke Scale with mild clinical grade classification if NIH Stroke Scale was 0-3, moderate clinical grade if NIH Stroke Scale was 4-15, and severe clinical grade if NIH Stroke Scale was 16-42. Clinical degrees were measured within 72 hours, 7^th days of onset and the 30^th days of onset. 59.4% of total subjects came up with moderate NIH Stroke Scale (4-15). On the 30^th day, as many as 85.9% of subjects had mild NIH Stroke Scale (0-3).

Spearman correlations test showed that levels of TGF-β1 which taken at 72 hours of onset was not significantly associated with clinical degrees on the 30th day. However, if scores of NIH Stroke Scales was classified into mild, moderate and severe and comparative T test was done then group with mild clinical degree at 30^th day had a significantly higher mean levels of TGF-β1 at ≤ 72 hours (mean 53467.82 ± 10834 pg/L) compared to group with moderate to severe clinical degree (mean 44277.67 ± 4560 pg / L) with significance p = 0.046. In addition, the results showed consistently negative correlation between the levels of TGF-β1 with clinical degrees of NIH Stroke Scales. TGF-β1 levels on the 7th dayon the 7th day of onset showed a negative correlation that was almost meaningless with NIH Stroke Scale scores at the 30^th days. (Table 3)

Table 3. Correlation of 72 hours and 7^th day biomarker levels to clinical degree on the 30th day

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Changes in 72 hours biomarker levels versus 7^th day biomarker levels might be categorized into "increasing" and "decreasing", then using t-test the differences between NIH Stroke Scale onon the 7th dayon the 7th day compared to 30^th days would be seen. Based on these categories, the groups with increased TGF-β1 levels had milder clinical degrees than the groups with decreased TGF-β1 levels (See Table 4). After the Spearman correlation test, there was no significant correlation between changes in the TGF-β1 levels at 72 hours and 7^th day observation to NIH Stroke Scale on the 30th day with r = 0.228 and p = 0.396.

Table 4. Mean NIH Stroke Scale values in biomarker levels group based on increase / decrease criteria

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Figure 1. The mean NIH Stroke Scale in the group has increased and decreased level of TGF-β1

4. Discussion

In this study, there were 64 subjects who met the inclusion criteria with mean age of 57.78 years. This finding was in accordance with aresearch on 2009 in Indonesia who had mean age for all cases of stroke were 58.8 years. Most risk factors were hypertension (93.75%), followed by dyslipidemia (88.33%) and smoking (73.44%). Other studies in Indonesia done by Kusuma (2009) and Yudiarto (2014) also showed that hypertension was the most risk factor, followed by diabetes and dyslipidemia. ^{[3, 13]} When viewed from the type of stroke, most patients (76.56%) in this study came up with a clinical syndrome of LACI. (Table 1)

TGFβ-1 was one of the cytokines that acted as anti-inflammatory and neurotrophic factor. TGFβ-1 plays a role in neuroplasticity of the brain to repair injured brain tissue post-stroke. On 2012 and 2013, Yoo Dobolyi showed that the TGFβ-1 had a neuroprotective effect, which suppresses inflammation and reduces infarct volume in experimental animals. ^[9] By using t-test, this study found that mean levels of TGF-β1 on the 30^th day in the group with mild clinical degree (mean 53467.82 ± 10834 pg / L) was significantly higher than in the group with the moderate-severe clinical degree (mean 44277.67 ± 4560 pg / L) with significance p = 0.046. Spearman correlation test results showed that levels of TGF-β1 at 72 hours of onset was not significantly associated with clinical degree on the 30^th day, but has consistently shown a negative correlation between the levels of TGF-β1 with clinical degrees NIHSS. TGF-β1 levels taken on the 7th dayon the 7th day of onset showed a negative correlation which almost meaningless with NIHSS score on the 30th day post-stroke (r = - 0.485 and p = 0057) (See Table 3). The results of this study supported the hypothesis of the role TGF-β1 as a protective factor and improve clinical outcomes post stroke.

A study done by Doyle in 2010 which using mice as a model for acute stroke showed an increased TGFβ signaling from the first day with peak on the 7.5^th day. The signal increasing was particularly visible on astrocytes. ^[14] However, previous studies mentioned that TGF-β1 serum levels in patients with acute stroke up to day 14 did not show a significant difference with the control. This study showed no significant difference between average levels of TGF-β1 at ≤ 72 hours and on the 7th day (p = 0384) with lower mean on the 7th day. This may be explained that the increaseing signal of TGFβ-1 in astrocytes at acute phase of stroke did not hav an impact to the increasing serum levels of TGFβ. It may take more than 7 days or more than 14 days to cause an increasing serum level of TGF-β1. So, the neuroprotective role of TGF-β1 was likely to be occured after the acute phase of stroke.

The weakness of this study was it included subjects with extensive infarct volume. NIH Stroke Scale severity on admission may influence the clinical outcomes on the 30th day after stroke. Therefore, we need further research with large number and more homogeneous samples. The role of physical exercise and psychological factors in post-stroke patients was also needed to be considered because it might affect the clinical outcome.

5. Conclusion

This study showed the average levels of TGFβ-1 taken at ≤ 72 hours post stroke were significantly higher in the group of subjects with mild NIHSS compared to moderate/severe NIHSS group on the 30th day post-stroke (p = 0.046). In the group of subjects who have elevated levels of TGFβ-1 post-stroke, the mean of NIHSS was lower (mean 1.14 ± 1.34) compared to the group of subjects with decreased levels of TGFβ-1 (mean 2.77 ± 3.3). It may support the hypothesis that TGFβ acts as a neuroprotective agent against brain tissue damage after ischemic stroke.

References

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