Grouping And Acclimatization Of Laboratory Animal Biology Essay

Male Swiss albino mice were produced from carnal experimental research lab, and used throughout the survey. They were housed in micro nylon boxes in a control environment ( temp 25±2 & A ; deg ; C ) and 12 hour dark /light rhythm with standard research lab diet and H2O ad libitum. The survey was conducted after obtaininginstitutional carnal ethicalcommittee clearance. As per the criterion pattern, the mice were segregated based on their gender and quarantined for 15 yearss before the beginning of the experiment. They were fed on healthy diet and maintained in hygienic environment in our carnal house.

Technique for Inducing Tumor

Assorted technique for initiation of malignant neoplastic disease in animate beings, viz, chemically induced ( utilizing 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 surveies of anticancer activity.

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In the present survey, cell lines induced malignant neoplastic disease in mice was used to measure the anticancer activity.

Evaluation OF ANTICANCER ACTIVITY

Initiation of malignant neoplastic disease utilizing DLA cells

Dalton ‘s Lymphoma ascites ( DLA ) cell was supplied by Amala malignant neoplastic disease research centre, Trissur, Kerala, India. The cells maintained in vivo in Swiss albino mice by intraperitoneal organ transplant. While transforming the tumour cells to the grouped animal the DLA cells were aspirated from peritoneal pit of the mice utilizing saline. The cell counts were done and farther dilution were made so that entire cell should be 1 ten 106, this dilution was given intraperitonealy.Let the tumour grow in the mice for minimal seven yearss before get downing interventions.

Treatment Protocol

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

Group- 1 served as the normal control.

Group- 2 served as the tumour 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 organic structure weight, Intraperitonealy. [ 7 ]

Group-4 Served as a intervention control group and was

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

Group-5 Served as a intervention control group and was

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

Treatment

In this survey, drug intervention was given after the 24 hour of vaccination, one time day-to-day for 14 yearss.

On twenty-four hours 14, after the last dosage, all mice from each group were sacrificed ; the blood was withdrawn from each mouse by ex post facto orbital rete method and the undermentioned parametric quantities were checked.

Hematologic parametric quantities

WBC count

RBC count

Hb content

Platelet count

Packed cell volume

2. Serum enzyme and lipid profile

Entire Cholesterol ( TC )

Triglycerides ( TG )

Aspartate amino Transferase ( AST )

Alanine amino Transferase ( ALT )

Alkaline Phosphatase ( ALP )

3. Derived parametric quantity

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 pit of each mouse was withdrawn by unfertile syringe and diluted with 0.8 milliliter of ice cold Normal saline or unfertile Phosphate Buffer Solution and 0.1 milliliter of tryphan blue ( 0.1 mg/ml ) and entire Numberss of the life cells were counted utilizing heamocytometer.

No of cells Dilution

Cell count = — — — — — — — — — — — — — — — — — — — — —

Area – Thickness of liquid movie

Hematologic parametric quantities

WBC count

RBC count

Platelet count

Hemoglobin

V ) Packed Cell Volume

I ) WBC count

The entire WBC count was found to be increased in malignant neoplastic disease control, when compared with normal and treated tumor-bearing mice. The entire WBC count were found to diminish significantly in animate beings treated with works infusion when compared with malignant neoplastic disease control, bespeaking that the anticancer nature of the infusion. [ 13 ]

two ) RBC and Hb

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

three ) Platelets

In Hodgkin lymphoma, increased in thrombocyte count frequently reported in research lab determination. Hence, I investigated this parametric quantity in the survey. [ 14 ]

four ) Packed cell volume

In any instance of anaemia the packed cell volume is lessenings.

SERUM ENZYME AND LIPID PROFILE

The serum was analyzed for the undermentioned parametric quantities

Aspartate amino Transferase ( AST )

Alanine amino Transferase ( ALT )

Alkaline Phosphatase ( ALP )

Entire Cholesterol ( TC )

Triglyceride ( TG )

1. Total CHOLESTEROL AND TRIGLYCERIDE ( lipid profile )

Abnormal blood lipoid profile has been associated with malignant neoplastic disease. In Hodgkin lymphoma, high cholesterin degree and low triglyceride degree has been reported. Hence I investigated this parametric quantity in the survey. [ 15 ]

LIVER ENZYMES ( AST, ALT, ALP ) .

Abnormal liver map seen in patient with Hodgkin lymphoma, [ 14 ] that these liver enzyme degrees markedly increase in tumour bearing mice. ALP is an enzyme chiefly derived from the liver, castanetss and in lesser sum from bowels, placenta, kidneys and leucocytes. An addition in ALP degrees in the serum is often associated with the assortment of disease. [ 16 ] ALP comprises a group of enzyme that catalyzes the phosphate esters in an alkaline environment, bring forthing an organic group and inorganic phosphate.

Markedly elevated serum ALP, hyperalakline-phosphatasemia, is seen preponderantly with more specific upsets ; including malignant bilious cirrhosis, hepatic lymphoma and sarcoidosis. [ 17 ] Hence, I investigated this parametric quantity in this survey.

DERIVED Parameters

1. Body weight:

All the mice were weighed, from the beginning to 15th twenty-four hours of the survey. Average addition in organic structure weight on the fifteenth twenty-four hours was determined.

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

% ILS was calculated by the undermentioned expression

Life span of treated group

% ILS =

All biochemical probes were done by utilizing COBAS MIRA PLUS-S Auto analyser from Roche Switzerland.

Hematologic trial are carried out in COBAS MICROS OT 18 from Roche.

Newly added High-tech instruments MAX MAT used for an car analyser for all biochemistry probes in blood sample.

Initiation of Cancer Using DLA Cell

Dalton ‘s Lymphoma Ascites ( DLA ) cells were supplied by Amala malignant neoplastic disease research centre, Trissur, Kerala, India. The cells were maintained in vivo in Swiss albino mice by intraperitoneal organ transplant. DLA cells aspirated from the peritoneal pit of mice were washed with saline and given intraperitonealy to develop carcinoma.

Consequence of EECS on Survival Time

Animals were divided into five groups of six animate beings each. Except the normal control group, the staying groups were inoculated with DLA cells ( 1×106 cells/mouse ) intraperitonealy on twenty-four hours ‘0 ‘ and intervention withEthanolic Extract of Coleus spicatus ( EECS ) started 24 hour after vaccination, at a dosage of 100mg and 200mg/kg/day. p.o. The normal and tumour control group was treated with same volume of 0.9 % Na chloride solution. All the interventions were given for 14 yearss. The addition in life span ( ILS ) of each group, dwelling of 6 mice was noted.

The antitumor efficaciousness of EECSwas compared with that of 5-fluorouracil ( Dabur pharmaceutical limited company India ; 5-FU, 20 mg/kg/day, i.p, for 14 yearss ) . The ILS of the treated groups was compared with that of the control group utilizing the undermentioned computation:

Addition in lifespan = [ ( T – C ) / C ] x 100

Where T = figure of yearss the treated animate being survived.

C = figure of yearss control animate beings survived.

Table No. 1-Effect of EECSon Hematological parametric quantities

Treatment

Entire WBC

Cells /mlx103

Rbc Count

Mill/cumm

Hemoglobin

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 average ± SEM for 6 animate beings in each group.

**a – Valuess are significantly different from Normal control ( G1 ) at P & A ; lt ; 0.001

*b – Valuess are significantly different from malignant neoplastic disease control ( G2 ) at P & A ; lt ; 0.01

**b – Valuess are significantly different from malignant neoplastic disease control ( G2 ) at P & A ; lt ; 0.001

Table No.2

Consequence of EECSon serum Enzymes and lipid proteins

Treatment

Cholesterol

( mg/dl )

TGL

( mg /dl )

AST

( U/L )

Elevation

( 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 average ± SEM for 6 animate beings 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 average ± SEM for 6 animate beings in each group.

**a – Valuess are significantly different from control ( G1 ) at P & A ; lt ; 0.001

*b – Valuess are significantly different from malignant neoplastic disease control ( G2 ) at P & A ; lt ; 0.01

**b – Valuess are significantly different from malignant neoplastic disease control ( G2 ) at P & A ; lt ; 0.001

Table No.3

Consequence of EECS on the life span, organic structure weight and malignant neoplastic disease cell count of tumour induced mice.

Treatment

Number of animate beings

% ILS Life span

Increase in Body weight gms

Cancer cell count

milliliter X 106

G1

6

& A ; gt ; 30 yearss

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 average ± SEM for 6 animate beings 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 average ± SEM for 6 animate beings in each group.

**a – Valuess are significantly different from normalcontrol ( G1 ) at P & A ; lt ; 0.001

*b – Valuess are significantly different from malignant neoplastic disease control ( G2 ) at P & A ; lt ; 0.01

**b – Valuess are significantly different from malignant neoplastic disease control ( G2 ) at P & A ; lt ; 0.001

RESULTS AND DISCUSSION

Consequence on Tumor Growth

In the DLA tumour control group, the mean life span of animate being was found to be 48 % where as EECS at the dosage of 100 and 200 mg/kg organic structure weight increase the life span to 70 % and 78 % severally. These values were important. However the mean life span of 5- FU intervention was found to be 90 % , bespeaking its potent antitumor nature. The anticancer nature ofEECS was evidenced by the important decrease in percent addition in organic structure weight of animate being treated with EECS at the dosage of 100 and 200 mg/kg organic structure weight when compared to DLA tumour bearing mice.

It was besides supported by the important decrease in jammed cell volume and feasible

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

Consequence 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 dosage of 100 and 200 mg/kg significantly increases the Hgb content, RBC, Platelets and significantly decreased the WBC count to about normal degree. All these consequences suggest the antineoplastic nature of the both dose ofEECS. However, the standard 5-FU at the dosage of 20 mg/kg organic structure weight produced better consequence in all these parametric quantities.

Consequence on Biochemical Parameters

The vaccination of DLA cellscaused significantly increase in the degree of Total Cholesterol, Aspartate amino Transferase, Alanine amino Transferase, Alkaline Phosphatase in the tumour control animate beings ( G2 ) , when compared to the normal group. The intervention with EECS at the dosage of 100 and 200 mg/kg organic structure weight reversed these alterations towards the normal degree. ( Table No. 2 ) All the value was found to be important. The intervention with standard 5- FU besides gave similar consequences.

Discussion

Coleus spicatus was traditionally used in the intervention of assorted unwellnesss. The present probe was carried out to measure the antitumor activity of EECS at the dosage of 100 and 200 mg/kg in DLA tumour bearing mice. The EECS at the dosage of 100 and 200 mg/kg significantly inhibited the tumour volume, packed cell volume, tumour ( feasible ) cell count and brought back the haematological parametric quantities to more or less normal degrees.

In DLA tumour bearing, a regular rapid addition in ascitic tumour volume was observed.Ascitic fluid is the direct nutritionary beginning for tumour cells and a rapid addition in ascitic fluid with tumour growing would be a agencies to run into the nutritionary demand of tumour cells. [ 18 ] Treatment with EECSinhibited the tumour volume, feasible tumour cell count and increased the life span of the tumour bearing mice. The dependable standards for judging the value of any antineoplastic drug are the protraction of the lifetime of animate beings. [ 19 ] It may be concluded that EECS by diminishing the nutritionary fluid volume and collaring the tumour growing increases the life span of DLA bearing mice. Therefore EECS have antitumor activity against DLA bearing mice.

Normally, in malignant neoplastic disease chemotherapy the major jobs that are being encountered are of myelo suppression and anaemia. [ 20,21 ] The anaemia encountered in tumour bearing mice is chiefly due to decrease in RBC or hemoglobin per centum and this may happen either due to press lack or due to haemolytic or myelopathic conditions. [ 22 ] Treatment with EECS brought back the haemoglobin ( Hb ) content, RBC and WBC count more or less to normal degrees significantly. This clearly indicates that EECS possess protective action on the hematopoietic system.

It was reported that the presence of tumour in the human organic structure or in the experimental animate beings is known to impact may work of the liver. The significantly elevated degree of entire cholesterin, TG, AST, ALT, ALP in serum of tumour inoculated carnal indicated liver harm and loss of functional unity of cell membrane. The important reversal of these alterations towards the normal by EECStreatments.

In the present survey, the biochemical scrutiny of DLA inoculated animate beings showed pronounced alterations bespeaking the toxic consequence of the tumour. The standardization of these effects observed in the serum treated with EECSsupported the powerful antitumor and hepatoprotective consequence 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

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Figure No.5

TREATMENT CONTROL ( G5 ) HIGH DOSES EECS

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