Effect of intra-arterial heparin flushing (IAHF) to prestin and vascular endothelial growth factor (VEGF) level in hearing loss patients

Elvita Rahmi Daulay; Delfitri Munir; Aznan Lelo; Terawan Agus Putranto; Aziza Ghanie Icksan; Tengku Siti Hajar Haryuna; Putri C. Eyanoer; Tina Christina L Tobing

doi:10.3897/pharmacia.70.e97501

Abstract

Background: According to the World Health Organization (WHO), hearing loss is the fourth largest disability globally, affecting an estimated 466 million people in 2018. In Indonesia, the prevalence of hearing loss was estimated at 16.8% in 2016. Intra-Arterial Heparin Flushing (IAHF) is an endovascular technique that uses heparin to promote reperfusion and increases Vascular Endothelial Growth Factor (VEGF) expression. VEGF is a polypeptide angiogenesis factor present in the nervous system, which functions as a neurotrophic and neuroprotective. Meanwhile, prestin is a protein of outer ear hair cells that shows early signs of hearing loss through increased levels in cases of ear hair cell damage.

Objective: This study aims to evaluate the effect of IAHF on prestin and VEGF levels in hearing-impaired patients.

Methods: The design is experimental with Pre-Post Test One-Group Only. A total of 22 patients with hearing loss were measured for prestin and VEGF before and 4 hours after the IAHF procedure.

Results: The results of the Wilcoxon test showed no significant differences in prestin level (p=0.658) and VEGF level (p=0.291) before and after the IAHF procedure. The mean showed an insignificant decrease in prestin level before and after the IAHF procedure with values of 1,185+1,229 pg/mL and 1,096+1,183 pg/mL, respectively. However, the VEGF level insignificantly increased before and after the procedure with values of 484.83+274.6 pg/mL and 498.79+257.7 pg/mL, respectively.

Conclusion: There were no significant differences in prestin and VEGF levels before and after the IAHF procedure. However, there was a decrease in the prestin level and an increase in the VEGF level.

Keywords

intra-arterial heparin flushing, Prestin, VEGF, hearing loss

Introduction

According to the World Health Organization (WHO), hearing disorder is the fourth largest disability globally, affecting 466 million people in 2018. In Indonesia, the prevalence of hearing disorder was estimated at 16.8% in 2016. Despite the high prevalence rate, most patients are unaware of their conditions, leading to inadequate treatment. However, only one-third of people with hearing disorder are aware of their problem (Djaali et al. 2018; WHO 2018; Michels et al. 2019).

Intra-Arterial Heparin Flushing (IAHF) is an endovascular technique that uses heparin to achieve reperfusion. Previous investigations have shown that IAHF, a modified technique of Digital Subtraction Angiography (DSA), can lead to clinical improvement for stroke patients in Gatot Soebroto Army Central Hospital. This condition can also increase Cerebral Blood Flow (CBF) in chronic ischemic stroke (Putranto et al. 2016a; Ratmono et al. 2016).

Heparin has been shown to release myeloperoxidase enzyme binding with endothelium, thereby increasing the bioavailability of Nitric Oxide (NO). The increase in NO can lead to high VEGF expression, which has been linked to improvement in hearing in animals with Noise-Induced Hearing Loss (NIHL) (Jozkowicz et al. 2001; Baldus et al. 2006; London and Gurgel 2014). VEGF is a factor of polypeptide angiogenic growth that is also present in the neuro system. It plays a crucial in the angiogenesis process and defense mechanism that can help supply oxygen and nutrition to ischemic brain tissue. VEGF also works directly with neurons to produce neurotrophic and neuroprotective effects. Some studies have also mentioned that VEGF induces axonal growth and neuron survival (Zhang et al. 2000; Jin et al. 2002; Yang et al. 2010) and plays a role in neurogenesis by inducing the release of neurotrophic factor (Thau-Zuchman et al. 2010; Nowacka and Obuchowicz 2013). An investigation in poultry showed the effect of VEGF to regenerate hair cells and supporting cells in the ear (Wan et al. 2020).

Prestin is a protein of outer ear hair cells that can be used as a biomarker to detect hearing loss. An increase in prestin levels indicates damage to the outer ear hair cell, which is an early sign of hearing loss (Tovi et al. 2019; Sun et al. 2020). Damaged hair cells released prestin serum into circulation. Studies have found increased prestin in Idiopathic Sudden Sensorineural Hearing Loss (ISSHL), which is identified as a marker of auditory hair cell integrity. Prestin is also recommended as prognostic to evaluate the effectiveness of treatment and improvement of hearing threshold in ISSHL (Naples et al. 2017; Tovi et al. 2019; Sun et al. 2020; Gomaa et al. 2021).

IAHF procedure has the potential to recover macro and micro circulation, and heparin also possesses an anti-oxidant attribute. Therefore, this study aims to determine the effect of IAHF on prestin and VEGF levels in patients with hearing loss. The results are expected to help the sensorineural hearing loss caused by decreased vascularization and endothelium dysfunction.

Material and method

Method

This experimental study was carried out using a Pre-Post Test One Group Only in the Cerebrovascular Center Gatot Soebroto Army Central Hospital Jakarta. The prestin and VEGF level was measured before and 4 hours after IAHF without any control group in Kramat Prodia Laboratory Clinic in Central Jakarta, from November 2021 to Mei 2022. This study has received approval from Ethical Committee for Health Research Universitas Sumatera Utara in the approval letter number 615/KEP/USU/2021. Based on the sample size calculation, the minimum sample was 21 participants. Consecutive sampling was used to gather all subjects, which was a type of non-probability sampling technique. All subjects were expected to fulfill the inclusion and exclusion criteria. The inclusion criteria were patients with sensorineural and mixed hearing loss, not pregnant, and those who agreed to sign informed consent, have prestin and VEGF level measurement. The exclusion criteria were patients with conductive type hearing loss, those with contrast allergy or heparin, blood clotting disorder, and a history of severe illness such as renal failure, cardiac decompensation, and malignancy.

Material

For IAHF, the fluoroscope Artis Q Zen produced by Siemens and a Terumo guide wire was used to reach the designated location. The materials used also included Heparin 5,000 IU by Inviclot, Normal Saline by Otsuka, Bethadine’s Povidon Iodin 10%, Alcohol 70%, lidocaine 2%, and Visipaque contrast produced by General Electric Healthcare in the United States. Human Prestin Enzyme–Linked Immunosorbent Assay (ELISA) Kit used was produced by MyBioSource in San Diego, California, United States, and VEGF ELISA Kit used was Quantikinie ELISA produced by Bio-Techne, Minneapolis, Minnesota, United States.

Study procedure

Before the IAHF procedure, patients with conductive type hearing loss or normal hearing were excluded through audiometry examination. To ensure safety, the potential side effects such as blood clotting were fully explained to the patients and their families. Subsequently, the patients were included in the IAHF procedure after they agreed to the possibility of side effects.

The IAHF procedure was carried out under sterile conditions. The equipment used included a catheter, introducer sheath, guide wire, lidocaine 2% in a 10 ml syringe, and mixed heparin 5,000 IU into 500 cc normal saline. Local anaesthesia was administered through lidocaine intracutaneous and subcutaneously. Access from the femoral artery was made with venous catheter 18G and the guide wire was inserted through fluoroscopy to ensure sure proper positioning. After removing the venous catheter, the introducer sheath was inserted, while its cap and guide wire was taken off simultaneously. Subsequently, the sheath was flushed with heparin to ensure there were no air bubbles. The angiography catheter was inserted using a guide wire until it reached the carotid artery. The angiography was performed using contrast Visipaque, while the carotid and vertebral arteries were flushed with 200 ml and 100 ml of heparin, respectively, at a pressure of 2 ml/s. The catheter was removed, followed by the sheath, and any bleeding was stopped by applying pressure with a gauze or using an angio seal.

The procedure for prestin and VEGF by ELISA method was carried out using prepared serum, then measured by spectrophotometer. Subsequently, prestin and VEGF levels were measured before and 4 hours after the IAHF procedure.

Data analysis

Data analysis in this study was carried out using SPSS (Statistical Program for Social Sciences) version 26. The normality of the data was determined by using the Shapiro-Wilk test, as the sample was expected to be <50. When the data were normally distributed, a paired t-test was used for hypothesis testing, otherwise, the Wilcoxon test was applied. Based on the statistical results, a significant difference between groups was indicated by a p-value less than 0.05, while a p-value higher than 0.05 indicated no difference.

Data availability

Data will be made available on request.

Results and discussion

Sample characteristics

This study involved 220 patients who passed through an audiometry examination. A total of 83 patients were in the normal hearing category, while 41 patients who had conductive-type hearing loss were excluded. After providing additional education and explanation about the procedure, 74 patients denied to participate and only 22 agreed to proceed. The differences among the patients were characterized and explained in Table 1.

Table 1.

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Characteristics of patients.

Category	N	%
Gender
Male	15	68.2
Female	7	31.8
Age
<60	7	31.8
≥60	15	68.2
Occupation
Civil Servant	1	4.5
Entrepreneur	13	59.1
Housewife	6	27.3
Unemployed	2	9.1
Comorbidities
1 Comorbidity	6	27.2
>1 Comorbidity	16	72.7
Type of hearing loss
Sensorineural	17	77.3
Mixed	5	22.7
Degree of hearing loss
Mild	10	45.5
Moderate	9	40.9
Moderate-severe	1	4.5
Severe	2	9.1
Very Severe	0	0

Out of the 22 patients, 15 (68.2%) were male, 15 (68.2%) were >60 years old, and the largest occupation group was entrepreneur, with 13 (59.1%). Furthermore, 16 (72.7%) of the patients have >1 comorbidity from various diseases such as hypertension, diabetic mellitus, dyslipidemia, stroke, and even COVID-19. The patient’s characteristics based on their degree of hearing loss were also categorized as presented in Table 2.

Table 2.

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Patient characteristics based on degree of hearing loss.

Characteristics	Degree of hearing loss
Characteristics	Mild	Moderate	Moderate-severe	Severe	Very severe
Gender
Male	8 (36.4%)	6 (27.3%)	0	1 (4.5%)	0
Female	2 (9.1%)	3 (13.6%)	1 (4.5%)	1 (4.5%)	0
Age
<60	3 (13.6%)	4 (18.2%)	0	0	0
≥60	7 (31.8%)	5 (22.7%)	1 (4.5%)	2 (9.1%)	0
Occupation
Civil servant	1 (4.5%)	0	0	0	0
Entrepreneur	6 (27.3%)	6 (27.3%)	1 (4.5%)	0	0
Housewife	2 (9.1%)	3 (13.6%)	0	1 (4.5%)	0
Unemployed	1 (4.5%)	0	0	1 (4.5%)	0
Comorbidities
1 Comorbidity	4 (18.2%)	2 (9.1%)	0	0	0
>1 Comorbidity	6 (27.3%)	7 (31.8%)	1 (4.5%)	2 (9.1%)	0

Prestin level analysis

Based on the normality data test in Table 3, the prestin level was not normally distributed, hence, the statistical analysis was continued using the Wilcoxon test.

Table 3.

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Analysis of the prestin level normality test.

	Prestin level (pg/mL)
	Mean±SD	Median (Min–Max)	P
Pre IAHF	1,185±1.229	519.52 (227–3.963)	0.000^a
4 hours Post IAHF	1,096±1.183	513.89 (239–3.069)	0.000^a

The Wilcoxon test showed that there was no significant difference in prestin level before and after the IAHF procedure, with p-value >0.05 (p=0.658; 95% CI), as shown in Table 4. Based on the mean, a decrease in prestin level before and after the IAHF procedure was obtained, with values of 1,185+1,229 pg/mL and 1,096+1,183 pg/mL, respectively. The graph of prestin level before and after the IAHF procedure can be seen in Fig. 1.

Figure 1.

Graph of prestin level before and after procedure.

Table 4.

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Analysis of prestin level before and after procedure.

	Prestin Level (pg/mL)
	Mean±SD	Median (Min–Max)	P
Pre IAHF	1,185±1,229	519.52 (227–3,963)	0.548^b
4 hours Post IAHF	1,096±1,183	513.89 (239–3,069)	0.548^b

VEGF level analysis

Based on the normality data test in Table 5, the VEGF level was not normally distributed, hence, statistical analysis was carried out using the Wilcoxon test.

Table 5.

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Analysis of the VEGF level normality test.

	VEGF Level (pg/mL)
	Mean±SD	Median (Min–Max)	P
Pre IAHF	484.83±274.6	485.35 (76–1,337)	0.023^a
4 hours Post IAHF	498.79±257.7	500.45 (86–1,220)	0.223^a

The Wilcoxon test showed no significant differences in VEGF level before and after the IAHF procedure, with a p-value of >0.05 (p=0.291; 95% CI), as shown in Table 6. However, the mean showed an increase in VEGF level before and after the IAHF procedure, with values of 484.83+274.6 pg/mL and 498.79+257.7 pg/mL, respectively. The graph of VEGF level before and after the IAHF procedure can be seen in Fig. 2.

Figure 2.

Graph of VEGF level before and after procedure.

Table 6.

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Analysis of VEGF level before and after procedure.

	VEGF Level (pg/mL)
	Mean±SD	Median (Min-Max)	P
Pre IAHF	484.83±274.6	485.35 (76–1,337)	0.291^b
4 hours Post IAHF	498.79±257.7	500.45 (86–1,220)	0.291^b

Discussion

IAHF was performed in several cases such as ischemic stroke, thrombotic cerebral vein, autism, and stenosis renal artery. Meanwhile, the IAHF procedure has been associated with an increase in CBF in the infarct area, as demonstrated by the Magnetic Resonance Imaging (MRI) before and after IAHF. This improvement in CBF resulted from the clot lysis and vasodilation that enhance macro and micro blood circulation and vasodilatation, rather than angiogenesis (Slevin et al. 2006; Angels Font et al. 2010; Perrey 2013; Putranto et al. 2016b, 2019; Ratmono et al. 2016; Pramono et al. 2019; Setiawan et al. 2019).

Heparin is an anti-coagulant that can be administered directly through IAHF. Based on previous investigations, heparin has several pharmacologic attributes such as anti-inflammation, anti-thrombotic, pro-fibrinolysis, anti-aggregative, anti-proliferation, anti-oxidant, and anti-ischemic (Shute et al. 2018; Pramono et al. 2019; Putra et al. 2019). The anti-oxidant effect involved the myeloperoxidase enzyme, produced by leukocytes to decrease NO levels by using it as the substrate to form free radicals. Heparin released myeloperoxidase enzyme binding with endothelium to increase NO bioavailability (Baldus et al. 2006).

Hair cell loss can occur happen through apoptotic or necrotic processes caused by cellular stress and ROS (Reactive Oxygen Species) (Peng and Jou 2010; Böttger and Schacht 2013; Dinh et al. 2015). Several reports have shown that the decrease in vascularization and endothelium dysfunction are pathogenic mechanisms for otologic disorders such as NIHL, presbycusis, and endolymphatic hydrops (Picciotti et al. 2004).

Prestin is a protein from outer ear hair cells that serves as a biomarker for assessing hearing function. Prestin concentrations increase in the presence of damaged outer ear hair cells. (C. Sun et al. 2020). It was also reported that VEGF in hearing improve in animals with noise-induced hearing loss on day-1 and day-7 after induction (London and Gurgel 2014).

Chadha et al. (2021) reported that more than 42% of people aged over 60 years experience hearing loss, and its prevalence increases with age (Chadha et al. 2021). In this study, a higher incidence of hearing loss was observed in patients aged >60 years old compared to those <60 years old, which was also consistent with the report of Shim et al. (2019). This indicated that an increase in age correlated with ear hair cell degeneration, supporting cell, as well as stria vascularis, and also induced nerve fiber decreased cochlear nerve. The central auditory nerve system also changed due to age increment in the auditory brain stem and auditory cortex (Fillit et al. 2016).

Study conducted by Gupta and friends showed that hearing loss prevalence was 2 times higher in males compared to females (Gupta et al. 2021), while others reported 1.1–1.43 times higher (Bansal et al. 2016). Similarly, this study obtained a hearing loss prevalence, with a comparison of 2.14:1 between males and females. This condition occurred because males were more probable to be exposed to noise in their workplace or recreational place. There was also a higher proportion of male smokers, which can lead to hearing loss because smoking has been linked to hearing loss. (Siegelaub et al. 1974). However, some studies reported that there is no correlation between gender and hearing loss in the non-industrial environment (Goycoolea et al. 1986; Helfer 2001).

There are several risk factors for hearing loss, namely inflammation process, systemic and genetic disease, as well as oxidative stress. Hearing loss had also been correlated with diabetes, cardiovascular such as increased blood pressure, and dyslipidemia (Sun et al. 2015).

Morbidity and mortality have also been correlated with hearing loss, primarily due to pathological mechanism of oxidative stress, vasoconstriction, and decreased blood flow that causes cochlear damage (Ohinata et al. 1994; Lee et al. 2001; Bamiou 2015; Oh et al. 2015; Rim et al. 2021).

In this study, patients with more than one comorbidity were more common compared to those with a single comorbidity. The comorbidities that were found included hypertension, diabetic mellitus, dyslipidemia, stroke, and COVID-19. This was similar to other investigations, where the incidence of hearing loss was in patients with metabolic syndrome (Aghazadeh-Attari et al. 2017; Shim et al. 2019).

This study showed that patients with sensorineural hearing loss were more compared to mixed type. Liu et al. discovered that the sensorineural type was the most common, followed by mixed, and conductive types. They also reported that most of the hearing loss degrees were mild and moderate, as confirmed by the dominant population in this study (Liu et al. 2011).

Prestin is produced by the cochlea and can be present in body circulation because it is small size enough to pass through the blood barrier. Prestin can be detected using ELISA in normal people as the result of outer ear hair changes (Parham and Dyhrfjeld-Johnsen 2016; Liba et al. 2017; Dogan et al. 2018; Parham et al. 2019; Tovi et al. 2019; Parker et al. 2022).

In this study, the prestin levels before and after the IAHF procedure was not significantly different but there was an 89 pg/mL mean difference. Based on previous investigations, prestin level was higher in ISSHL patients compared to those with non-hearing loss. However, there was no significant association between prestin levels before the IAHF procedure and hearing threshold improvement. (Sun et al. 2020). Hana et al. discovered that in 300 NIHL patients, there was a significantly decreased prestin level before and after therapy, with values of 169+88.4 pg/mL and 114+99.2 pg/mL, and a mean difference of 55 pg/mL (Hana and Bawi 2018).

The lack of insignificant results in this study can be due to factors outside the scope of outer ear hair cells, such as spiral ganglion dysfunction (Frisina et al.). Furthermore, prestin only showed outer ear hair cell condition and the IAHF procedure focused on oxidative stress treatment.

VEGF has a protective effect on neuron cells from hypoxia and ROS increment (Clinkard et al. 2013). London Gurgel (2014) also found that VEGF is beneficial as a therapy for sensorineural hearing loss (London and Gurgel 2014). In this study, VEGF measurement was carried out before and after the IAHF procedure. The results showed no significant difference between the two stages but the mean level was increased. The use of heparin as an agent in IAHF had been proven to increase NO bioavailability (Baldus et al. 2006) and induce VEGF expression (Jozkowicz et al. 2001). Furthermore, based on the use of the human umbilical vein, heparin increased VEGF expression in the first 24 hours by increasing Hypoxia Inducible Factors-1 (HIF-1) and AP-1 (Fazil et al. 2021).

An increased VEGF level has been associated with treatment in animal models of NIHL (Picciotti et al. 2005; Picciotti et al. 2006). Another report showed that VEGF inhibitor induced hearing loss due to decreased local blood flow and microvascular thrombosis in the labyrinth artery (Cheng et al. 2019).

The various VEGF levels obtained in this study might be due to influences from hypoxia, differentiation cell process, growth factor, and oncogenic (Kolch et al. 1995). The results showed that patients with comorbidity such as metabolic syndrome can have lower VEGF compared to normal people. According to Barylski et al. (2008), the more comorbidities a patient has, the lower the levels of initial VEGF (Barylski et al. 2008).

Conclusion

This study showed that most of the patients were over 60 years old and had mild to moderate hearing loss. The results indicated that the IAHF procedure in sensorineural and mixed-type hearing loss reduced prestin levels and increase VEGF, although the changes were not statistically significant. Therefore, future study should investigate the use of the IAHF procedure to treat hearing loss caused by endothelial dysfunction and vascular abnormalities in sensorineural and mixed-type cases.

Funding

This study did not receive any specific grant.

Declaration of interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by Universitas Sumatera Utara and PT. Prodia Widyahusada Tbk.

References

Aghazadeh-Attari J, Mansorian B, Mirza-Aghazadeh-Attari M, Ahmadzadeh J, Mohebbi I (2017) Association between metabolic syndrome and sensorineural hearing loss: A cross-sectional study of 11,114 participants. Diabetes, Metabolic Syndrome and Obesity 10: 459–465. https://doi.org/10.2147/DMSO.S150893

Angels Font M, Arboix A, Krupinski J (2010) Angiogenesis, neurogenesis and neuroplasticity in ischemic stroke. Current Cardiology Reviews 6(3): 238–244. https://doi.org/10.2174/157340310791658802

Baldus S, Rudolph V, Roiss M, Ito WD, Rudolph TK, Eiserich JP, Sydow K, Lau D, Szöcs K, Klinke A, Kubala L, Berglund L, Schrepfer S, Deuse T, Haddad M, Risius T, Klemm H, Reichenspurner HC, Meinertz T, Heitzer T (2006) Heparins increase endothelial nitric oxide bioavailability by liberating vessel-immobilized myeloperoxidase. Circulation 113(15): 1871–1878. https://doi.org/10.1161/CIRCULATIONAHA.105.590083

Bamiou DE (2015) Chapter 35 - Hearing disorders in stroke. In: Aminoff MJ, Boller F, Swaab CN (Eds) The human auditory system. Elsevier, vol. 129, 633–647. https://doi.org/10.1016/B978-0-444-62630-1.00035-4

Bansal D, Varshney S, Malhotra M, Joshi P, Kumar N (2016) Unilateral sensorineural hearing loss: A retrospective study. Indian Journal of Otology 22(4): 262–267. https://doi.org/10.4103/0971-7749.192174

Barylski M, Kowalczyk E, Banach M, Ciećwierz J, Pawlicki L, Kowalski J (2008) Plasma total antioxidant activity in comparison with plasma NO and VEGF levels in patients with metabolic syndrome. Angiology 60(1): 87–92. https://doi.org/10.1177/0003319708327165

Böttger EC, Schacht J (2013) The mitochondrion: A perpetrator of acquired hearing loss. Hearing Research 303: 12–19. https://doi.org/10.1016/j.heares.2013.01.006

Chadha S, Kamenov K, Cieza A (2021) The world report on hearing. Bulletin of the World Health Organization 99(4): 242–242A. https://doi.org/10.2471/BLT.21.285643

Cheng J, Wang L, Zhu LN, Wang L (2019) Bilateral sensorineural hearing loss induced by regorafenib. Journal of Clinical Pharmacy and Therapeutics 44(6): 963–965. https://doi.org/10.1111/jcpt.13009

Clinkard D, Amoodi H, Kandasamy T, Grewal AS, Chen S, Qian W, Chen JM, Harrison RV, Lin VYW (2013) Changes in expression of vascular Endothelial growth factor and its receptors in the aging mouse Cochlea, part 1: The normal-hearing mouse. Journal of Otolaryngology 2013: 6. https://doi.org/10.1155/2013/430625

Dinh CT, Goncalves S, Bas E, Van De Water TR, Zine A (2015) Molecular regulation of auditory hair cell death and approaches to protect sensory receptor cells and/or stimulate repair following acoustic trauma. Frontiers in Cellular Neuroscience 9(March): 1–15. https://doi.org/10.3389/fncel.2015.00096

Djaali N, Putri S, Sihite D, Endarti A, Hastono S, Amqam H (2018) Analysis of the relationship of noise exposure and hearing loss in PPSU workers in North Jakarta. Science Journal of Public Health 6(5): 140–144. https://doi.org/10.11648/j.sjph.20180605.14

Dogan M, Sahin M, Cetin N, Yilmaz M, Demirci B (2018) Utilizing prestin as a predictive marker for the early detection of outer hair cell damage. American Journal of Otolaryngology 39(5): 594–598. https://doi.org/10.1016/j.amjoto.2018.07.007

Fazil S, Shah H, Noreen M, Yar M, Farooq Khan A, Zaman Safi S, Yousef Alomar S, Fahad Alkhuriji A, Mualla Alharbi H, Sohail Afzal M (2021) Evaluation of molecular mechanisms of heparin-induced angiogenesis, in human umbilical vein endothelial cells. Journal of King Saud University. Science 33(6): 101534. https://doi.org/10.1016/j.jksus.2021.101534

Fillit HM, Rockwood K, Young JB (2016) Brocklehurst’s Textbook of Geriatric Medicine and Gerontology. Elsevier Health Sciences. https://books.google.co.id/books?id=dowtDAAAQBAJ

Frisina RD, Ding B, Zhu X, Walton JP (2016) Age-related hearing loss: Prevention of threshold declines, cell loss and apoptosis in spiral ganglion neurons. Aging 8(9): 2081–2099. https://doi.org/10.18632/aging.101045

Gomaa NA, Jimoh Z, Campbell S, Zenke JK, Szczepek AJ (2021) Biomarkers for inner ear disorders: Scoping review on the role of biomarkers in hearing and balance disorders. Diagnostics 11(1): 1–19. https://doi.org/10.3390/diagnostics11010042

Goycoolea MV, Goycoolea HG, Rodriguez LG, Farfan CR, Martinez GC, Vidal R (1986) Effect of life in industrialized societies on hearing in natives of easter island. The Laryngoscope 96(12): 1391–1396. https://doi.org/10.1288/00005537-198612000-00015

Gupta K, Varshney S, Tyagi AK, Kumar A, Sood R (2021) Epidemiological study of unilateral sensorineural hearing loss in adults. Indian Journal of Otology 27(2): 96–100. https://doi.org/10.4103/indianjotol.INDIANJOTOL

Hana R, Bawi B (2018) Prestin, otolin-1 regulation, and human 8-oxoG DNA glycosylase 1 gene polymorphisms in noise-induced hearing loss. Ibnosina Journal of Medicine and Biomedical Sciences 10(02): 60–64. https://doi.org/10.4103/ijmbs.ijmbs_4_18

Helfer K (2001) Gender, age, and hearing. Seminars in Hearing 22: 271–286. https://doi.org/10.1055/s-2001-15631

Jin K, Zhu Y, Sun Y, Mao XO, Xie L, Greenberg DA (2002) Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo. Proceedings of the National Academy of Sciences of the United States of America 99(18): 11946–11950. https://doi.org/10.1073/pnas.182296499

Jozkowicz A, Cooke JP, Guevara I, Huk I, Funovics P, Pachinger O, Weidinger F, Dulak J (2001) Genetic augmentation of nitric oxide synthase increases the vascular generation of VEGF. Cardiovascular Research 51(4): 773–783. https://doi.org/10.1016/s0008-6363(01)00344-3

Kolch W, Martiny-Baron G, Kieser A, Marmé D (1995) Regulation of the expression of the VEGF/VPS and its receptors: Role in tumor angiogenesis. Breast Cancer Research and Treatment 36(2): 139–155. https://doi.org/10.1007/BF00666036

Lee H, Whitman GT, Lim JG, Lee SD, Park YC (2001) Bilateral sudden deafness as a prodrome of anterior inferior cerebellar artery infarction. Archives of Neurology 58(8): 1287–1289. https://doi.org/10.1001/archneur.58.8.1287

Liba B, Naples J, Bezyk E, Campbell C, Mei M, Parham K (2017) Changes in serum prestin concentration after exposure to cisplatin. Otology & Neurotology 38(10): e501–e505. https://doi.org/10.1097/MAO.0000000000001616

Liu H, Zhang H, Bentler RA, Mo L, Han D, Zhang L (2011) Audiometric records analysis in a clinical population in China. ORL. Journal for Oto-Rhino-Laryngology and Its Related Specialties 73(5): 237–245. https://doi.org/10.1159/000330041

London NR, Gurgel RK (2014) The role of vascular endothelial growth factor and vascular stability in diseases of the ear. The Laryngoscope 124(8): E340–E346. https://doi.org/10.1002/lary.24564

Michels TC, Duffy MT, Rogers DJ (2019) Hearing loss in adults: Differential diagnosis and treatment. American Family Physician 100(2): 98–108.

Naples J, Cox R, Bonaiuto G, Parham K (2017) Prestin as an Otologic Biomarker of Cisplatin Ototoxicity in a Guinea Pig Model. Otolaryngology - Head and Neck Surgery 158(3): 541–546. https://doi.org/10.1177/0194599817742093

Nowacka M, Obuchowicz E (2013) BDNF and VEGF in the pathogenesis of stress-induced affective diseases: An insight from experimental studies. Pharmacological Reports 65(3): 535–546. https://doi.org/10.1016/s1734-1140(13)71031-4

Oh I-H, Lee JH, Park DC, Kim M, Chung JH, Kim SH, Yeo SG (2015) Hearing loss as a function of aging and diabetes mellitus: A cross sectional study. PLoS ONE 9(12): e116161. https://doi.org/10.1371/journal.pone.0116161

Ohinata Y, Makimoto K, Kawakami M, Haginomori S-I, Araki M, Takahashi H (1994) Blood Viscosity and Plasma Viscosity in Patients with Sudden Deafness. Acta Oto-Laryngologica 114(6): 601–607. https://doi.org/10.3109/00016489409126112

Parham K, Dyhrfjeld-Johnsen J (2016) Outer Hair Cell Molecular Protein, Prestin, as a Serum Biomarker for Hearing Loss: Proof of Concept. Otology & Neurotology 37(9): 1217–1222. https://doi.org/10.1097/MAO.0000000000001164

Parham K, Sohal M, Petremann M, Romanet C, Broussy A, Tran Van Ba C, Dyhrfjeld-Johnsen J (2019) Noise-induced trauma produces a temporal pattern of change in blood levels of the outer hair cell biomarker prestin. Hearing Research 371: 98–104. https://doi.org/10.1016/j.heares.2018.11.013

Parker A, Parham K, Skoe E (2022) Noise exposure levels predict blood levels of the inner ear protein prestin. Scientific Reports 12(1): 1154. https://doi.org/10.1038/s41598-022-05131-z

Peng T-I, Jou M-J (2010) Oxidative stress caused by mitochondrial calcium overload. Annals of the New York Academy of Sciences 1201(1): 183–188. https://doi.org/10.1111/j.1749-6632.2010.05634.x

Perrey S (2013) Promoting Motor Function by Exercising the Brain. Brain Sciences 3(1): 101–122. https://doi.org/10.3390/brainsci3010101

Picciotti PM, Torsello A, Cantore I, Stigliano E, Paludetti G, Wolf FI (2005) Expression of vascular endothelial growth factor and its receptors in the cochlea of various experimental animals. Acta Oto-Laryngologica 125(11): 1152–1157. https://doi.org/10.1080/00016480510044241

Picciotti P, Torsello AI, Wolf F, Paludetti G, Gaetani E, Pola R (2004) Age-dependent modifications of expression level of VEGF and its receptors in the inner ear. Experimental Gerontology 39(8): 1253–1258. https://doi.org/10.1016/j.exger.2004.06.003

Picciotti PM, Fetoni AR, Paludetti G, Wolf FI, Torsello A, Troiani D, Ferraresi A, Pola R, Sergi B (2006) Vascular endothelial growth factor (VEGF) expression in noise-induced hearing loss. Hearing Research 214(1): 76–83. https://doi.org/10.1016/j.heares.2006.02.004

Pramono A, Soetikno R, Kartamihardja AHS, Maskoen TT, Putranto TA, Setiawan E (2019) Short insight of pediatric cerebral venous thrombosis and the safety of intra-arterial heparin flushing as a new therapeutic method. Bali Medical Journal 8(2): 560. https://doi.org/10.15562/bmj.v8i2.1463

Putra B, Jonny E, Soewandi AH (2019) Renal intra-arterial heparin flushing for blood pressure control in refractory hypertension: A new method to salvage the unsalvageable renal artery stenosis. Indian Journal of Nephrology 29: 200–203. https://doi.org/10.4103/ijn.IJN_194_18

Putranto TA, Yusuf I, Murtala B, Wijaya A (2016a) Intra arterial heparin flushing increases cereberal blood flow in chronic ischemic stroke patients. The Indonesian Biomedical Journal 8(2): 119. https://doi.org/10.18585/inabj.v8i2.204

Putranto T, Irawan Y, Murtala B, Wijaya A (2016b) Intra arterial heparin flushing increases manual muscle test – Medical Research Councils (MMT-MRC) score in chronic ischemic stroke patient. Bali Medical Journal (Fort Sam Houston, Tex.) 5: 25. https://doi.org/10.15562/bmj.v5i2.200

Putranto TA, Ratmono T, Yusuf I, Murtala B, Wijaya A (2019) Intra arterial heparin flushing effect on motor evoked potentials in chronic ischemic stroke patients. Bali Medical Journal 8(2): 540. https://doi.org/10.15562/bmj.v8i2.1493

Ratmono T, Patellongi I, Kaelan C, Wijaya A, Asadul A (2016) L-glutamate profile in acute ischemic stroke after intra arterial heparin flushing. Bali Medical Journal 5(1): 11. https://doi.org/10.15562/bmj.v5i1.211

Rim H-S, Kim M-G, Park D-C, Kim S-S, Kang D-W, Kim S-H, Yeo S-G (2021) Association of Metabolic Syndrome with Sensorineural Hearing Loss. Journal of Clinical Medicine 10(21): 4866. https://doi.org/10.3390/jcm10214866

Setiawan E, Soetikno R, Risan NA, Shelly S, Ratmono T, Putranto TA, Pramono A (2019) Measuring motor evoked-potentials in children with autism spectrum disorders accompanied with cerebral vein thrombosis following intraarterial heparin flushing. Bali Medical Journal 8(2): 617. https://doi.org/10.15562/bmj.v8i2.1531

Shim HS, Shin HJ, Kim MG, Kim JS, Jung SY, Kim SH, Yeo SG (2019) Metabolic syndrome is associated with hearing disturbance. Acta Oto-Laryngologica 139(1): 42–47. https://doi.org/10.1080/00016489.2018.1539515

Shute JK, Puxeddu E, Calzetta L (2018) Therapeutic use of heparin and derivatives beyond anticoagulation in patients with bronchial asthma or COPD. Current Opinion in Pharmacology 40: 39–45. https://doi.org/10.1016/j.coph.2018.01.006

Siegelaub AB, Friedman GD, Adour K, Seltzer CC (1974) Hearing Loss in Adults. Archives of Environmental Health: An International Journal 29(2): 107–109. https://doi.org/10.1080/00039896.1974.10666542

Slevin M, Kumar P, Gaffney J, Kumar S, Krupinski J (2006) Can angiogenesis be exploited to improve stroke outcome? Mechanisms and therapeutic potential. Clinical Science 111(3): 171–183. https://doi.org/10.1042/CS20060049

Sun Y-S, Fang W-H, Kao T-W, Yang H-F, Peng T-C, Wu L-W, Chang Y-W, Chou C-Y, Chen W-L (2015) Components of metabolic syndrome as risk factors for hearing threshold shifts. PLoS ONE 10(8): e0134388. https://doi.org/10.1371/journal.pone.0134388

Sun C, Xuan X, Zhou Z, Yuan Y, Xue F (2020) A preliminary report on the investigation of prestin as a biomarker for idiopathic sudden sensorineural hearing loss. Ear, Nose, and Throat Journal 99(8): 528–531. https://doi.org/10.1177/0145561319849949

Thau-Zuchman O, Shohami E, Alexandrovich A, Leker RR (2010) Vascular endothelial growth factor increases neurogenesis after traumatic brain injury. Journal of Cerebral Blood Flow and Metabolism 30: 1008–1016. https://doi.org/10.1038/jcbfm.2009.271

Tovi H, Ovadia H, Eliashar R, de Jong MA, Gross M (2019) Prestin autoantibodies screening in idiopathic sudden sensorineural hearing loss. European Annals of Otorhinolaryngology, Head and Neck Diseases 136(2): 99–101. https://doi.org/10.1016/j.anorl.2017.11.013

Wan L, Lovett M, Warchol ME, Stone JS (2020) Vascular endothelial growth factor is required for regeneration of auditory hair cells in the avian inner ear. Hearing Research 385: 107839. https://doi.org/10.1016/j.heares.2019.107839

WHO [World Health Organization] (2018) Addressing the rising prevalence of hearing loss. World Health Organization. https://apps.who.int/iris/handle/10665/260336

Yang L, Lu Z, Ma X, Cao Y, Sun L-Q (2010) A therapeutic approach to nasopharyngeal carcinomas by DNAzymes targeting EBV LMP-1 gene. Molecules 15(9): 6127–6139. https://doi.org/10.3390/molecules15096127

Zhang ZG, Zhang L, Jiang Q, Zhang R, Davies K, Powers C, Van Bruggen N, Chopp M (2000) VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain. The Journal of Clinical Investigation 106(7): 829–838. https://doi.org/10.1172/JCI9369