CHAPTER 3

MATERIAL AND METHODS OF THE RESEARCH

3.1 Place and Time of Research

This research has been conducted for a month from January to February

2016. The research has been done at the Laboratory of Animals Model

Department of Biochemistry at Medicine Faculty of Universitas Airlangga for the

treatment of experimental animals. Tissue processing and histopathology scoring

of rat kidney has been made in Laboratory of Veterinary Pathology Department

of Veterinary Pathology at Veterinary Medicine Faculty of Universitas Airlangga.

3.2 Material and Equipment of Research

3.2.1 Material of Research

3.2.1.1 Experimental Animal

Healthy 3 months old male Wistar rats (Rattus norvegicus) with 150-200

grams average weight were used in this research. The rats were fed standard diet

and water and maintained at the same place.

3.2.1.2 Spirulina platensis

Spirulina platensis was obtained from CV. Mutiara Tasnim, Sleman,

Yogyakarta, which produces a pure breed of Spirulina platensis in powder form.

Spirulina powder has been extracted using ethanol in order to gain the active

substances, then Spirulina suspension will be made by mixing spirulina extract

35
 36

with 0.5% CMC Na and aquadest. The dosages of Spirulina platensis extract

used were 200 mg/kg BW, 400 mg/kg BW, and 800 mg/kg BW based on the

research that has been done before by Sharma et al. (2007).

3.2.1.3 Ethanol

Ethanol in this research is used as animal model material to injury kidney.

Ethanol with the dosages of 50% concentration, 10ml/kg BW was determined

from the research by Gunawan (2010) and preliminary research.

3.2.1.4 Chemical Material

The chemical used include 96% ethanol for material extraction, 0.5% CMC

for extract solvent, 10% formalin for tissue fixation, Hematoxylin and Eosin

(HE) staining, paraffin, 30%, 50%, 70%, 80%, 90%, 100% concentration of

alcohol, and xylol absolute.

3.2.2 Equipment of Research

The equipment used to extract Spirulina platensis were rotary evaporator,

rotary microtome, water bath, mortir, stamper, sterile tube, and digital scales.

The equipment used for sample preparation were enclosure plastic box (36

x 28 x 12) cm with wire cover, drinking places, oral sonde, spuit, and dissection

set (forceps, scalpel, blade, surgical scissors).

The equipment used for histopathology slide preparation were object glass,

pipette, tissue processor automatic, water bath, hot plate, microtome, and blade.

Meanwhile, equipment used for histopathology scoring was microscope

(Olympus CX-21).
 37

3.3 Method of Research

3.3.1 Spirulina platensis Extraction Using Maceration Method

Spirulina platensis in 3000 grams of powder was extracted by maceration

process using ethanol 96%. In this process, Spirulina platensis powder was

placed in a stoppered container with the solvent (ethanol) and allowed to stand at

room temperature for seven days with frequent agitation until the soluble matter

has dissolved. The mixture then was strained, the marc (the damp solid material)

was pressed, and the combined liquids were clarified by subsidence or filtration.

The extracts were evaporated and concentrated using rotary evaporator after

filtration (Singh, 2008).

3.3.2 Ethanol Residue Identification in Spirulina Extract

The purpose of ethanol identification on ethanol extract of Spirulina is to

ensure that there is no ethanol residue in Spirulina extract result. Ethanol

identification in Spirulina extract was done with esterification test by diluting the

Spirulina extract in aquadest then it is added by using CH 3COOH and

concentrated with H2SO4, then it was heated. If the identification ethanol result

shows positive, it means there is specific ester smell in Spirulina extract.

(Depkes, 1977).
 38

3.3.3 Rat Treated

The next stage of this research was the treatment in male rats (Rattus

norvegicus) which has been held in a cage trial. 20 male Wistar rats in 3 months

old with 150-200 grams average weight was reared in Laboratory of Animals

Model Department of Biochemistry at Medicine Faculty of Universitas

Airlangga, randomized by a lottery and were divided into five groups, and then

adapted to the environment for one week. In the second week of experiment,

animals were treated respectively for twenty one days, seven days of ethanol

treatment followed by fourteen days of Spirulina platensis treatment. Animals

were fasted for 3 hours before treated with ethanol. Feeding and drinking is ad

libitum.

3.3.4 Number of Samples

Minimum sample size are eligible for analysis determined formula

premises Federer (Federer, 1967) as follows:

t (n-1) 15

5 (n-1) 15

n4

Specification:

n = number of samples

t = number of treatment
 39

Through the formula above, the results is n 4. This shows the minimum

sample size is 4. In this study, four samples were used for each experiment group

(n = 4).

In this study, researchers used five groups of samples, among others:

Negative Control : Rats were administered with 0.5% CMC Na 10 ml/kg

BW per orally for 21 days. Ethanol and Spirulina

platensis were not be administered.

Positive Control : Rats were administered with 50% ethanol with a

dosage of 10 ml/kg BW per orally for 7 days, then

continued by giving 0.5% CMC 10 ml/kg BW per

orally for 14 days. Spirulina platensis was not be

administered.

Treatment 1 : Rats were administered with 50% ethanol with a

dosage of 10 ml/kg BW per orally for 7 days, then

continued by giving Spirulina platensis extract with a

dosage of 200 mg/kg BW per orally for 14 days.

Treatment 2 : Rats were administered with 50% ethanol with a

dosage of 10 ml/kg BW per orally for 7 days, then

continued by giving Spirulina platensis extract with a

dosage of 400 mg/kg BW per orally for 14 days.

 40

Treatment 3 : Rats were administered with 50% ethanol with a

dosage of 10 ml/kg BW per orally for 7 days, then

continued by giving Spirulina platensis extract with a

dosage of 800 mg/kg BW per orally for 14 days.

3.3.5 Organ Samples Collection

At the end of the treatment, food was withdrawn from the rats and they

were fasted overnight but the animals had free access to water. They were then

euthanized through chloroform inhalation. Dissection using a dissecting set was

done to isolate both of dexter and sinister rat kidneys that located in the

abdominal cavity of primary retroperitoneal left and right vertebralis column

(Setiadi, 2007). Thereafter, the kidneys were suspended in 10% formalin for

fixation preparation in histological processing.

3.3.6 Making the Slide Preparation

The procedure to make histopathology slide are tissue fixation, washing

(Laundering), tissue processing, dehydration, clearing, impregnation, embedding,

tissue cutting, and stained the histopathology anatomy slides.

3.4 Experimental Design

This study included experimental research with Complete Random Design

pattern. Using 5 groups, two control groups and three treatment groups are

chosen with simple randomization method of sampling. Each unit treatment is

repeated four times. The inspection process was carried out by observation and
 41

scoring of the rat kidney injury. Assessment was done by comparing the results

observed in the treatment groups and control, as well as between the treatment

groups.

C- CMC Na O1

AM R
C Ethanol CMC Na O2
+

T1 Ethanol Spirulina O3

T2 Ethanol Spirulina O4

T3 Ethanol Spirulina O5

AM = Animal Models

R = Randomization

CMC Na = 0.5% CMC Na in aquadest 10 ml/kg BW

Ethanol = 50% Ethanol 10 ml/kg BW for making the rats kidney injury

Spirulina = Spirulina platensis extract suspension

C- = Negative control group using 0.5% CMC Na 10 ml/kg BW for 21

days. Ethanol and Spirulina platensis will not be administered.

 42

C+ = Positive control group using 50% Ethanol 10 ml/kg BW for 7 days,

then 0,5% CMC 10 ml/kg BW for 14 days prior. Spirulina platensis
will not be administered.

T1 = Treatment for group 1 using 50% ethanol 10 ml/kg BW for 7 days

(started from 8th day 14th day) and Spirulina platensis with a dosage
200 mg/kg BW for 14 days (started from 15th day 28th day).

T2 = Treatment for group 2 using 50% ethanol 10 ml/kg BW for 7 days

(started from 8th day 14th day) and Spirulina platensis with a dosage
400 mg/kg BW for 14 days (started from 15th day 28th day).

T3 = Treatment for group 3 using 50% ethanol 10 ml/kg BW for 7 days

(started from 8th day 14th day) and Spirulina platensis with a dosage
800 mg/kg BW for 14 days (started from 15th day 28th day).

O1-5 = Observation and scoring of rat kidney.

3.5 Research Variable

3.5.1 Independent Variables

The independent variable in this study is the dosages of Spirulina platensis

extract 200, 400, and 800 mg/kg BW (Sharma et al., 2007) and dosages of

ethanol (50% concentration, 10ml/kg BW) (Gunawan, 2010).

3.5.2 Dependent Variables

The dependent variable in this study is the histopathology score of kidney.

3.5.3 Control Variable

 43

Control variables in this study are the, rat species, feed and drink, rat ages,

enclosures and tools used for research.

3.6 Operational Definition of Variables

Histopathological changes in kidney was found by observing the

microscopic level changes in the kidney of experimental animals. The

observation method in this research is used the modification of The Banff 97

scoring method for kidney organ damage (Racusen et al., 1999). The kidney

histopathological changes aspects were observed as congestion, hemorrhage,

degeneration, and necrosis. Congestion of glomerulus is the accumulation of

erythrocytes in the glomerular capillary and cause glomerular space narrowing.

Congestion was observed in 10 glomerulus using 100x and 400x magnification

(swelling of glomerulus or congestion and Bowmans space narrowing).

Hemorrhage is the accumulation of erythrocytes in the renal interstitial tissue.

Degeneration of kidney tubular epithelial cells is the swelling of kidney tubular

epithelial cells thus narrowing the lumen of the kidney tubules. Necrosis of

kidney tubular epithelial cells is the cell death characterized by nucleus that look

denser and darker (pyknosis) or nucleus lysis and form a spread chromatin

fragments (karyorrhexis) or nucleus cell disappearance (karyolysis).

 44

Hemorrhage, degeneration, and necrosis were observed in 5 visual fields using

400x magnification (Azmijah et al., 2010).

3.7 Grading Techniques

Table 3.1 The Scoring of Kidney Histopathology Appearance (Racusen et al.,

1999).

Kerusakan Skor Perubahan Histopatologi

yang diamati
0 1 dari 10 glomerulus terjadi kongesti.
1 2 to 3 glomerulus terjadi kongesti.

Kongesti 2 4 to 6 glomerulus terjadi kongesti.

3 > 6 glomerulus terjadi kongesti.

0 Tidak ada infiltrasi eritrosit interstisial.

1 < 25% infiltrasi eritrosit interstisial.

Hemorrhagi
2 26 50% infiltrasi eritrosit interstisial.

3 > 50% infiltrasi eritrosit interstisial.

0 Tidak terdapat degenerasi pada sel epitel tubulus ginjal.

1 < 25% terdapat degenerasi pada sel epitel tubulus ginjal.

Degenerasi
2 26 50% terdapat degenerasi pada sel epitel tubulus ginjal.

3 > 50% terdapat degenerasi pada sel epitel tubulus ginjal.

 45

0 Tidak terdapat nekrosis pada sel epitel tubulus ginjal.

Nekrosis 1 < 25% terdapat nekrosis pada sel epitel tubulus ginjal.

2 26 50% terdapat nekrosis pada sel epitel tubulus ginjal.

3 > 50% terdapat nekrosis pada sel epitel tubulus ginjal

3.8 Processing and Data Analysis

Data were obtained in the form of score level overview of histopathological

changes in the rat kidney are arranged in table to be analyzed with Kruskal Wallis

test. The degree of change is processed by ranking method and if noticeable

change were observed, data analyses were continued using Mann-Whitney test

(Kusriningrum, 2012). The entire analysis is done using computer statistic

program SPSS 18.0 for Windows.

 46

3.9 Research Flowchart

20 Wistar Albino Rats, 3 months old

150 - 200 grams

Adapted for 7 days after

randomization

C- C+ T1 T2 T3
4 Wistar Rats 4 Wistar Rats 4 Wistar Rats 4 Wistar Rats 4 Wistar Rats

Kidney damage process

(started from the 8th day the 14th day)

C- C+ T1 T2 T3
0.5% CMC Na 50% Ethanol 50% Ethanol 50% Ethanol 50% Ethanol
in aquadest 10 10ml/kg BW 10ml/kg BW 10ml/kg BW 10ml/kg BW
ml/kg BW each each rat 7 days each rat 7 days each rat 7 days each rat 7 days
rat 7 days orally orally orally orally orally

Treatment
(started from the 15th day the 28th day)

C- C+ T1 T2 T3
0.5% CMC Na 0.5% CMC Na Spirulina Spirulina Spirulina
in aquadest 10 in aquadest 10 platensis extract platensis extract platensis extract
ml/kg BW each ml/kg BW each 200 mg/kg BW 400 mg/kg BW 800 mg/kg BW
rat 14 days rat 14 days each rat 14 days each rat 14 days each rat 14 days
orally orally orally orally orally

Euthanized by chloroform inhalation on the 29th day

Rat kidney collection on the 29th day

Tissue process

Scoring

Data Analysis