1. BACKGROUND:

The development of science and technology gives good and bad impact for human. The good impact of the development of medical technology is that one of them is in the field of molecular biology and medical science. Currently, stem cells in the field of molecular biology or immunology engineering stem cell development in the treatment of cell cortical organs in human adversely affects badly to the technology which generated noise.¹

The value of noise threshold above 80 decibels is considered safe for the labors who work 8 hours/day or 40 hours/week in the factory letter circular of the Minister of worker, Transmigration and Cooperatives No. SE-01/MEN/1978, the value of threshold for noise at work is the highest intensity and the mean value is still acceptable to the labor without causing the loss of ability of hearing for the continues time more than 8 hours a day or 40 hours a week.¹

According to the Occupational Safety & Health Organization (WHO), people with hearing disorder are 360 millions (5.3%), of which 328 millions (91%) are mostly adults and 32 million (9%) are suffered by children. The rate of hearing disorder is directly proportional to the rate of age.²

The damaging cell mitochondria internally can be caused by cycle of ROS that can release the Apaf 1 so that Hsp 70 penetration to the membrane mitochondrialwhich causes the releasing of Cytochrome C and Caspase 3. The death of this cell begins with translocation Cytochrome C to activate Caspase 3 then finally the cell is damaging and having apoptosis of tissue cochlear. Noise exposure and heat continuously can lead to expression of Caspase 3, Caspase 8 and Caspase 9 and trigger Caspase 3 as the death executor through Programme cell death. Another study on cohlear tissue, evaluating the results of the immunohistochemical HSP70 examination and SEM ultrastructural cochlear examination that obtained 50% damaging on the cortical organ cells caused by noise 80-110 dB.^3,4

a. Early Research:

This study wasabout the effectiveness of cell culture in stage I and followed by stage II for immunohistochemical examination.

b. Research Stage

This research was conducted in 2 stages:

a. Stage I study: the examination of cell culture membrane basilaris from cortical organ by:

• Taking of basilar cell membrane culture on the cochlea with dissecting microscope.

• Gaining the several influence of good cell organ cortical which would be cultured.

b. Stage II study: Immunohistochemical examination

• Knowing serum Ki67 and Hsp 70 on the organs of the cortic cochlear

• Obtaining cell cultures on the membrane basilar of the cochlear cortical organ.

• Calculating the amount of damaging cell on the membrane basilaris of the cortic organ.

2. RESEARCH METHOD:

The rats were kept in a plastic cage with the size of 50x20x10 cm covered by wire netting. The enclosure of the cage is coated with 0,5-1cm thick husk rice and replaced every three days. The room light was controlled for 12 light hours (6 am to 6 pm) and 12 dark hours (6pm to 6 am), meanwhile the temperature and the humidity of the room was let in the natural range. The rat food came from PT. Charon Pokpan CP 551 type and the water was supplied excessively everyday.

a. Study Sample:

The samples of the experimental animals were male wistar white rats with 7-9 weeks of age obtained from the Biology Maternity of USU and examined sample preparation by cell culture with the correct procedure.

b. Maintenance and Adaptation:

A total of 25 male white rats were kept in plastic cage with the size of 50x20x10 cm covered by wire netting. The enclosure of the cage is coated with 0,5-1cm thick husk rice and replaced every three days. The room light was controlled for 12 light hours (6 am to 6 pm) and 12 dark hours (6pm to 6 am), meanwhile the temperature and the humidity of the room was let in the natural range. Animals try to be given free access to food and drink.

c. Organ of Corti extraction procedure:

Euthanasia of rats was done with the proper method (appropriate method with correct procedure). The head and neck deflection of rats was by using ethanol 70% after that did decapitation to the head of rats with surgical scissor and cut subcutaneous on rats skin that covered to expose the skull bone. Then, cut the skull in midsagital to two parts by cutting the median line to the eye area.After that, the cut was done with sagittal way on the temporal bone to the part of the vertebral canal until it reaches the olfactory bulb. The temporal piece was taken out circularly until the part of basal of the cochlea and apex then cut (sliced) with microtome of 2 μm. Samples were put in a 6 cm diameter of petri cup before examinationimmunohistochemical and cell culture cochlear were done (Ilyas, 2008).⁴

d. Immunohistochemistry examination:

Cochlear was fixated with formalin 10% for 24 hours and classified with formic acid for 4 days. Then preparat was made of by⁴:

· Dehydration with alcohol series ranging from 30,50,60,70,80,90,96% each 1hour.

· Put into 3x1 hour absolute alcohol.

· Cleansed in alcohol and xylol at a ratio of 1: 3, 1: 1 and 1: 3 each for 1 hour.

· Soaked in 24 hours of xylol. Put in xylol and paraffin with a ratio of 3:1. 1:1 and 1:3 respectively for 1 hour in an incubator temperature of 58 ° C.

· Cochlear implantation in paraffin has been melted in the paraffin box and left at room temperature for 24 hours.

· Paraffin blocks which in the cochlea was cut with microtom thickness of 6μm.

· The preparat which had provided were stained with HE.

Figure 1. The piece of cochlea in sagittal view, basilar membrane was identified

3. RESULTS:

Table 1 The result value of Ki67 measurement

group	N	mean	SD	Min-Max	P
P0	5	0.09	0.009	0.071 – 0.093	<0.001
P1	5	0.41	0.07	0.326 – 0.481
P2	5	0.21	0.03	0.190 – 0.255
P3	5	0.11	0.01	0.101 – 0.121
P4	5	0.13	0.02	0.108 – 0.052
P5	5	0.1	0.01	0.083 – 0.108

The result of analysis by using test Anova showed that there was significant difference mean (p<0.001) for the levelKI 67 between group P0 to the group P5. The lowest mean KI 67 was found in the group rats control with mean 0.09 (SD=0.009) and the highest group rats which given treatment noise 80-110 dB with mean 0.41 (SD=0.07).

Tabel 2 The result value of HSP 70 ELISA measurement

Group	N	Mean	SD	Min-Max	P
P0	5	2.79	0.43	2.12 – 3.10	<0.001
P1	5	17.08	3.22	13.55 – 20.50
P2	5	8.47	1.20	7.45 – 10.37
P3	5	3.95	0.44	3.46 – 4.36
P4	5	4.92	1.09	3.78 – 6.11
P5	5	3.44	0.46	2.66 – 3.78

The result of analysis by using test Anova showed that there was significant difference mean (p<0.001) for the levelHSP 70between group P0 to the group P5. The lowest meanHSP 70was found in the group rats control with mean 2,79 (SD=0.043) and the highest group rats which given treatment noise 80-110 dB with mean17,08 (SD=3.22).

Table 3 The correlation Pearson KI 67 dan HSP 70

Group	N	P	R	Interpretation
P0	5	<0.001	+1	Very strong
P1	5	<0.001	+1	Very strong
P2	5	<0.001	+1	Very strong
P3	5	<0.001	+1	Very strong
P4	5	<0.001	+1	Very strong
P5	5	<0.001	+1	Very strong

The result of analysis by using test correlation pearson showed that Ki67 had the significant positive correlation which very strong (r=1) with HSP-70. All the group pf rats showed that the similar result. The group PO to the group P5 had the significant relation <0.001 (very strong)

4. CONCLUSION AND DISCUSSION:

the result of analysis showed that Ki67 was significant to the PO-P5 with rats control (PO) with mean 0.09(SD=0.009) and the highest in the group rats which given treatment noise (P5) with mean 0.41(SD=0.07).

The result of ELISA by usingAnova test was found that there was difference mean P<0.01 to the group PO (not given noise) to the group P5 (given noise 80-110dB). The lowest mean HSP 70 was found that group rat control (PO) with mean 2.79(SD=0.43) and the highest in the groups rats which given noise 80-110 dB with mean 17.08(SD=3.22).

The test correlation of pearson showed that Ki67 had significant positive correlation which strongly (r=1) with HSP 70. All the group of rats showed that the similar result in the study which strongly related to apoptosis in the internal mitochondria.^3,4,6

REFERENCE:

1. Bashiruddin, J.Pencegahan Gangguan Pendengaran, tantangan dan harapan. dalam implementasi program sound hearing 2030. Pidato Pengukuhan guru besar THT-KL FK UI, Jakarta. 2010.

2. Kemenkes. KementerianKesehatanRepublik Indonesia. Pendengaran Sehatuntuk Hidup Bahagia. Jakarta. 2013.Available from: http://www.depkes.go.id/index.php?vw=2&id=2245. [Accesed 20 March 2017].

3. Hu, B.H., Cai, Q., Manohar, S., et al. Differential expression of apoptosis-related genes in the cochlea of noise-exposed rats. Neuroscience. 2009:161(3), pp. 915-925.

4. Ilyas, S. Pendekatan Kasus Mekanisme Apoptosis oleh Signal Intrinsik, Ekstrinsik dan Faktor Pemicu Apoptosis dalam Jurnal Ilmu dan Budaya vol. 28. Jakarta Selatan: Universitas Nasional. 2008:35-8

5. Makishima T., Hochman L., Armstrong, P, et al. Inner ear dysfunction in caspase-3 deficient. BMC Neuroscience. 2011:12(102), pp. 1-9.

6. Herwanto.Y. Basshiruddin. J , Ilyas S., Lubis D .N. Relationship Hsp 70 with Noisy Intensity of the the Heat Shock Response in : Expression of Caspase 3 and Apoptosis in Cochlear Rattus Norvegicus.In: Abstract Book 6^thAsia Pacific Otolgy,Neurootology and Skull Base Conference. 5^th Asean Academy of NeuroOtology & Audilogy (AANOA) Congress. 6 ^thMalaysianInternational ORL-HNS Congress & 34^th Annulal General Meeting of the MSO-HNS, 29^{th –}31^st 2014, Hotel Equatorial Penang Malaysia. 2014:p.83.

7. Babu, H., Cheung, G., Kettenmann, H., Palmer, T.D., Kempermann, G., 2007.Enriched monolayer precursor cell cultures from micro-dissected adult mouse dentate gyrus yield functional granule cell-like neurons. PloS ONE, 2, e388.

8. Babu, H., Claasen, J.H.,Kannan, S., Runker, A.E., Palmer, T. And Kempermann, G., 2011. A protocol for isolation and enriched monolayer cultivation of neural precursor cells from mouse dentate gyrus. Frontiers in Neuroscience, 5(89), pp. 1-10.

9. Dhingra, P.L., 2010. Anatomy of the Ear : Peripheral Receptors and Physiology of Auditory and Vestibular System. In: Dhingra, P.L., Diseases of the Ear, Nose and Throat 5th Edition. India: Elsevier, pp. 17-8

10. Gacek,R.R., 2009. Anatomy of Auditory and Vestibular System. In :Snow Jr, J.B. and Wackym P.A., Malengger’s Otorhinolaryngology Head and Neck Surgery 17th Edition. Philadelphia: People’sMedical Publshing House, p. 157l.

11. Pujol, R., Pickett, S.B., Nguyen, T.B. and Stone, J.S., 2014. Large basolateral processes on type II hari cells are novel processing units in mammalian vestibular organs. The Journal of Comparative Neurology, 522, pp. 3141-3159.

12. Saputra, V., 2006. Dasar-dasar stem cell danpotensiaplikasinyapadailmukedokteran. Cermin Dunia Kedokteran, 153, pp. 21-25

Received on 25.09.2018 Accepted on 06.10.2018

Asian J. Pharm. Res. 2019; 9(1): 19-21.

DOI: 10.5958/2231-5691.2019.00004.2