Grouping And Acclimatization Of Laboratory Animal Biology Essay

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Male Swiss albino mice were produced from animal experimental laboratory, and used throughout the study. They were housed in micro nylon boxes in a control environment (temp 25±2°C) and 12 hrs dark /light cycle with standard laboratory diet and water ad libitum. The study was conducted after obtaininginstitutional animal ethicalcommittee clearance. As per the standard practice, the mice were segregated based on their gender and quarantined for 15 days before the commencement of the experiment. They were fed on healthy diet and maintained in hygienic environment in our animal house.

Technique for Inducing Tumor

Various technique for induction of cancer in animals, viz, chemically induced (using DMBA/croton oil, etc)[3] virus induced, cell line induced (sarcoma - 180, ULCA fibro sarcoma and Jensen sarcoma, mouse lung fibroblast cells L-929, Dalton's Lymphoma Ascites (DLA), Ehrlich Ascites Carcinoma (EAC)[4,5,6]methods have been used in experimental studies of anticancer activity.

In the present study, cell lines induced cancer in mice was used to evaluate the anticancer activity.

EVALUATION OF ANTICANCER ACTIVITY

Induction of cancer using DLA cells

Dalton's Lymphoma ascites (DLA) cell was supplied by Amala cancer research center, Trissur, Kerala, India. The cells maintained in vivo in Swiss albino mice by intraperitoneal transplantation. While transforming the tumor cells to the grouped animal the DLA cells were aspirated from peritoneal cavity of the mice using saline. The cell counts were done and further dilution were made so that total cell should be 1 x 106, this dilution was given intraperitonealy.Let the tumor grow in the mice for minimum seven days before starting treatments.

Treatment Protocol

Swiss Albino mice were divided in to five group of six each. All the animals in four groups were injected with DLA cells (1 x 106 cells per mouse) intraperitonealy, and the remaining one group is normal control group.

Group- 1 served as the normal control.

Group- 2 served as the tumor control. Group 1 and 2 receives normal diet and Water.

Group- 3 served as the positive control, was treated with injection fluorouracil at 20 mg/kg body weight, Intraperitonealy. [7]

Group-4 Served as a treatment control group and was

Administered Ethanolic Extract of Coleus spicatus (EECS) at a low dose of 100mg/kg through orally. (ED50value)

Group-5 Served as a treatment control group and was

Administered Ethanolic Extract of Coleus spicatus (EECS) at a High dose of 200mg/kg through orally.(ED50value)

Treatment

In this study, drug treatment was given after the 24 hrs of inoculation, once daily for 14 days.

On day 14, after the last dose, all mice from each group were sacrificed; the blood was withdrawn from each mouse by retro orbital plexus method and the following parameters were checked.

Hematological parameters

WBC count

RBC count

Hb content

Platelet count

Packed cell volume

2. Serum enzyme and lipid profile

Total Cholesterol (TC)

Triglycerides (TG)

Aspartate amino Transferase (AST)

Alanine amino Transferase (ALT)

Alkaline Phosphatase (ALP)

3. Derived parameter

Body weight

Life span (%)

h. Cancer Cell Count

EVALUATION OF CLINICAL PARAMETERS [8, 9, 10, 11]

Cancer cell count[12]

The fluid (0.1ml) from the peritoneal cavity of each mouse was withdrawn by sterile syringe and diluted with 0.8 ml of ice cold Normal saline or sterile Phosphate Buffer Solution and 0.1 ml of tryphan blue (0.1 mg/ml) and total numbers of the living cells were counted using heamocytometer.

No of cells Dilution

Cell count = ------------------------------------------

Area - Thickness of liquid film

Hematological parameters

WBC count

RBC count

Platelet count

Hemoglobin

v) Packed Cell Volume

i) WBC count

The total WBC count was found to be increased in cancer control, when compared with normal and treated tumor-bearing mice. The total WBC count were found to decrease significantly in animals treated with plant extract when compared with cancer control, indicating that the antitumor nature of the extract.[13]

ii) RBC and Hb

RBC and Hb content decreases with tumor bearing mice when compared with Normal control mice.

iii) Platelets

In Hodgkin lymphoma, increased in platelet count often reported in laboratory finding. Hence, I investigated this parameter in the study.[14]

iv) Packed cell volume

In any case of anemia the packed cell volume is decreases.

SERUM ENZYME AND LIPID PROFILE

The serum was analyzed for the following parameters

Aspartate amino Transferase (AST)

Alanine amino Transferase (ALT)

Alkaline Phosphatase (ALP)

Total Cholesterol (TC)

Triglyceride (TG)

1. TOTAL CHOLESTEROL AND TRIGLYCERIDE (lipid profile)

Abnormal blood lipid profile has been associated with cancer. In Hodgkin lymphoma, high cholesterol level and low triglyceride level has been reported. Hence I investigated this parameter in the study.[15]

LIVER ENZYMES (AST, ALT, ALP).

Abnormal liver function seen in patient with Hodgkin lymphoma, [14] that these liver enzyme levels markedly increase in tumor bearing mice. ALP is an enzyme mainly derived from the liver, bones and in lesser amount from intestines, placenta, kidneys and leukocytes. An increase in ALP levels in the serum is frequently associated with the variety of disease. [16] ALP comprises a group of enzyme that catalyzes the phosphate esters in an alkaline environment, generating an organic radical and inorganic phosphate.

Markedly elevated serum ALP, hyperalakline-phosphatasemia, is seen predominantly with more specific disorders; including malignant biliary cirrhosis, hepatic lymphoma and sarcoidosis.[17]Hence, I investigated this parameter in this study.

DERIVED PARAMETERS

1. Body weight:

All the mice were weighed, from the beginning to 15th day of the study. Average increase in body weight on the 15th day was determined.

2. Percentage increase in life span (ILS) [13]

% ILS was calculated by the following formulae

Life span of treated group

%ILS =

All biochemical investigations were done by using COBAS MIRA PLUS-S Auto analyzer from Roche Switzerland.

Hematological test are carried out in COBAS MICROS OT 18 from Roche.

Newly added Hi-Tech instruments MAX MAT used for an auto analyzer for all biochemistry investigations in blood sample.

Induction of Cancer Using DLA Cell

Dalton's Lymphoma Ascites (DLA) cells were supplied by Amala cancer research center, Trissur, Kerala, India. The cells were maintained in vivo in Swiss albino mice by intraperitoneal transplantation. DLA cells aspirated from the peritoneal cavity of mice were washed with saline and given intraperitonealy to develop carcinoma.

Effect of EECS on Survival Time

Animals were divided into five groups of six animals each. Except the normal control group, the remaining groups were inoculated with DLA cells (1x106 cells/mouse) intraperitonealy on day '0' and treatment withEthanolic Extract of Coleus spicatus (EECS)started 24 hrs after inoculation, at a dose of 100mg and 200mg/kg/day. p.o. The normal and tumor control group was treated with same volume of 0.9% sodium chloride solution. All the treatments were given for fourteen days. The increase in life span (ILS) of each group, consisting of 6 mice was noted.

The antitumor efficacy of EECSwas compared with that of 5-fluorouracil (Dabur pharmaceutical ltd. India; 5-FU, 20 mg/kg/day, i.p, for 14 days). The ILS of the treated groups was compared with that of the control group using the following calculation:

Increase in lifespan = [(T - C) / C] x 100

Where T = number of days the treated animal survived.

C = number of days control animals survived.

Table No. 1-Effect of EECSon Hematological parameters

TREATMENT

Total WBC

Cells /mlx103

Rbc Count

Mill/cumm

Hb

Gm/dl

PCV %

Platelets

Lakhs/cumm

G1

10.05 ±1.45

4.05±0.95

12.30 ±2.25

14.45±2.60

3.05±0.80

G2

14.65 ±2.60a**

2.65±0.55a**

7.30 ±1.04a**

31.45±4.55a**

1.70±0.48a**

G3

11.78 ±1.92b**

3.85±0.90b**

11.6 ±1.75b**

19.25±2.40b**

2.68±0.65b**

G4

13.40±2.15b*

3.05±0.68b*

9.45±1.40b*

25.22±3.85b*

1.98±0.28b*

G5

12.50 ±2.25b**

3.35±0.80b**

10.80±1.85b**

21.30±2.25b**

2.22 ±0.48b**

G1 - Normal Control, G2 - Cancer Control, G3 - Positive control, G4 - Treatment control (low dose EECS), G5 - Treatment control (High dose EECS)

All values are expressed as mean ± SEM for 6 animals in each group.

**a - Values are significantly different from Normal control (G1) at P < 0.001

*b - Values are significantly different from cancer control (G2) at P < 0.01

**b - Values are significantly different from cancer control (G2) at P < 0.001

Table No.2

Effect of EECSon serum Enzymes and lipid proteins

Treatment

Cholesterol

(mg/dl)

TGL

(mg /dl)

AST

(U/L)

ALT

(U/L)

ALP

(U/L)

G1

100.20±3.62

125.8±4.45

38.50 ±1.28

38.40 ±1.40

128.45 ±2.25

G2

142.90±4.64a**

208.20±6.78a**

88.6±2.75a**

61.33±2.40a**

240.65±4.30a**

G3

110.40±3.92b**

157.35±3.80b**

56.30 ±1.95b**

42.40±1.75b**

165.25±2.32b**

G4

127.65±4.44b*

188.94±3.75b*

78.45 ±2.33b*

56.40±2.25b*

202.35±3.40b*

G5

121.30±3.85b**

170.65±3.28b**

71.52±1.60b**

44.40 ±1.55b**

190.45±2.55b**

All values are expressed as mean ± SEM for 6 animals in each group.

G1 - Normal Control, G2 -Cancer Control, G3 -Positive control, G4 -Treatment control (low dose EECS),

G5- Treatment control (High dose EECS).

All values are expressed as mean ± SEM for 6 animals in each group.

**a - Values are significantly different from control (G1) at P < 0.001

*b - Values are significantly different from cancer control (G2) at P < 0.01

**b - Values are significantly different from cancer control (G2) at P < 0.001

Table No.3

Effect of EECS on the life span, body weight and cancer cell count of tumor induced mice.

Treatment

Number of animals

% ILS Life span

Increase in Body weight grams

Cancer cell count

ml X 106

G1

6

>30 days

2.30±0.52

-

G2

6

48%

7.78±1.24a**

2.45±0.30a**

G3

6

90%

3.44±0.65b**

1.40±0.42b**

G4

6

70%

6.50±1.15b*

1.85±0.55b*

G5

6

78%

5.60±0.95b**

1.64±0.42b**

All values are expressed as mean ± SEM for 6 animals in each group.

G1 - Normal Control, G2 -Cancer Control, G3 -Positive control, G4 -Treatment control (low dose EECS),G5- Treatment control (High dose EECS).

All values are expressed as mean ± SEM for 6 animals in each group.

**a - Values are significantly different from normalcontrol (G1) at P < 0.001

*b - Values are significantly different from cancer control (G2) at P < 0.01

**b - Values are significantly different from cancer control (G2) at P < 0.001

RESULTS AND DISCUSSION

Effect on Tumor Growth

In the DLA tumor control group, the average life span of animal was found to be 48% where as EECS at the dose of 100 and 200 mg/kg body weight increase the life span to 70% and 78% respectively. These values were significant. However the average life span of 5- FU treatment was found to be 90%, indicating its potent antitumor nature. The antitumor nature ofEECS was evidenced by the significant reduction in percent increase in body weight of animal treated with EECS at the dose of 100 and 200 mg/kg body weight when compared to DLA tumor bearing mice.

It was also supported by the significant reduction in packed cell volume and viable

Tumor cell count in both the extent of treatment when compared to the DLA tumor control. (Table No .3)

Effect on Hematological Parameters

As shown in (Table No.1) RBC, Hgb, Platelets were decreased and WBC count was significantly increased in the DLA control group compared to the normal control group. Treatment with EECS at the dose of 100 and 200 mg/kg significantly increases the Hgb content, RBC, Platelets and significantly decreased the WBC count to about normal level. All these results suggest the anticancer nature of the both dose ofEECS. However, the standard 5-FU at the dose of 20 mg/kg body weight produced better result in all these parameters.

Effect on Biochemical Parameters

The inoculation of DLA cellscaused significantly increase in the level of Total Cholesterol, Aspartate amino Transferase, Alanine amino Transferase, Alkaline Phosphatase in the tumor control animals(G2), when compared to the normal group. The treatment with EECS at the dose of 100 and 200 mg/kg body weight reversed these changes towards the normal level. (Table No. 2) All the value was found to be significant. The treatment with standard 5- FU also gave similar results.

DISCUSSION

Coleus spicatus was traditionally used in the treatment of various illnesses. The present investigation was carried out to evaluate the antitumor activity of EECS at the dose of 100 and 200 mg/kg in DLA tumor bearing mice. The EECS at the dose of 100 and 200 mg/kg significantly inhibited the tumor volume, packed cell volume, tumor (viable) cell count and brought back the hematological parameters to more or less normal levels.

In DLA tumor bearing, a regular rapid increase in ascitic tumor volume was observed.Ascitic fluid is the direct nutritional source for tumor cells and a rapid increase in ascitic fluid with tumor growth would be a means to meet the nutritional requirement of tumor cells. [18] Treatment with EECSinhibited the tumor volume, viable tumor cell count and increased the life span of the tumor bearing mice. The reliable criteria for judging the value of any anticancer drug are the prolongation of the lifespan of animals.[19] It may be concluded that EECS by decreasing the nutritional fluid volume and arresting the tumor growth increases the life span of DLA bearing mice. Thus EECS have antitumor activity against DLA bearing mice.

Usually, in cancer chemotherapy the major problems that are being encountered are of myelo suppression and anemia. [20,21] The anemia encountered in tumor bearing mice is mainly due to reduction in RBC or hemoglobin percentage and this may occur either due to iron deficiency or due to hemolytic or myelopathic conditions.[22] Treatment with EECS brought back the hemoglobin (Hb) content, RBC and WBC count more or less to normal levels significantly. This clearly indicates that EECS possess protective action on the haemopoietic system.

It was reported that the presence of tumor in the human body or in the experimental animals is known to affect may function of the liver. The significantly elevated level of total cholesterol, TG, AST, ALT, ALP in serum of tumor inoculated animal indicated liver damage and loss of functional integrity of cell membrane. The significant reversal of these changes towards the normal by EECStreatments.

In the present study, the biochemical examination of DLA inoculated animals showed marked changes indicating the toxic effect of the tumor. The normalization of these effects observed in the serum treated with EECSsupported the potent antitumor and hepatoprotective effect of theEECS.

VIABLE CELL COUNT

Figure No .1

NORMAL CONTROL

Fig2

Figure No .2

CANCER CONTROL

Fig-1

Figure No.3

POSITIVE CONTROL

Fig3

Figure No4

TREATMENT CONTROL (G4) LOW DOSE EECS

P3240215

Figure No.5

TREATMENT CONTROL (G5) HIGH DOSES EECS

P3240216

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