An Apparatus for the determination of rat paw Edema during In vivo Evaluation of Anti-inflammatory agents

 

U. S. Jijith1,2, S. Jayakumari1

1School of Pharmaceutical Sciences, Vels Institute of Sciences, Technology and Advanced Studies

(VISTAS), Pallavaram, Chennai-600117, India

2College of Pharmaceutical Sciences, Govt. Medical College, Kozhikode-673008, Kerala, India

*Corresponding Author E-mail: nisajayaa@gmail.com

 

ABSTRACT:

In vivo evaluation of anti-inflammatory agents normally involves the measurement of rat paw volume. Some notable equipment for in vivo measurement of rat paw volume includes plethysmometer, screw gauge and vernier calipers. Balance method is also reported for the purpose. The present study describes development and validation of a simple apparatus for the determination of rat paw volume suitable for in vivo evaluation of anti-inflammatory agents.The calibration plot obtained with the developed apparatus was linear with a high R2 value of 0.983. Thus it was confirmed that the apparatus is useful for the determination of rat paw volume.

 

KEYWORDS: Paw edema, plethysmometer, screw gauge, vernier calipers, balance method.

 

 


1. INTRODUCTION:

Evaluation of anti-inflammatory activity is a common requirement in development of novel therapeutic agents against inflammation [1-6]. A good number of methods have been suggested for determination of rat paw edema during in vivo evaluation of anti-inflammatory agents [7-10]. Some notable equipment includes plethysmometer [11], screw gauge [12] and vernier calipers [13]. Balance method is also reported. Among these, the plethysmometer enjoys a prime position in its acceptability for measuring paw edema [14,15]. Modified apparatus are also reported [16]. It has already been reported that measurement of raw paw edema is always prone to error. In order to develop a cheap, reliable and simple laboratory method, we have developed an apparatus for the determination of rat paw edema.

 

2. MATERIALS AND METHODS:

Materials:

Purified water was used in the study.

 

Design and development of an apparatus for determination of rat paw volume:

In order to develop a cheap, reliable and simple laboratory method, we have developed an apparatus for the determination of rat paw edema. The instrument works on the principle of displacement of water on immersion of rat paw into a chamber filled with water [17,18].

 

Working of the apparatus:

The apparatus consists of a T-tube. One of the openings of the main tube (not the opening of the side tube) of the T-tube is closed and that part is firmly fixed onto a heavy base. The fixing of the T-tube to the heavy base provides sufficient stability to the apparatus during its operation. The position of the T-tube was in such a way that the side tube with opening faces to one side of the apparatus. The other end of the main tube was faced upward and allows entry of rat paw. The side tube is fitted with a side pipe. When water is added into the apparatus it gets filled into the apparatus until the level reaches the point of attachment of the side pipe.  Now after filling the apparatus with water, if we add some more water it will flow outwards through the side tube. Interestingly, the volume expelled through the side tube would be equal to the volume of water added. Thus we presumed that if we could immerse the rat paw into the apparatus pre-filled with water, it would displace water. Moreover, the volume of water displaced by the paw would be equal to its volume. The larger diameter of the main tube provides easy visibility and immersion of rat paw through it.

 

Calibration of the apparatus:

In order to ensure that the developed apparatus works properly and accurately, we carried out calibration of the apparatus. The calibration was carried out by addition of standard volume of water into the prefilled chamber of the apparatus. The water displaced through the outlet was collected into a pre-weighed microcentrifuge tube. The final weight of the microcentrifuge tube with displaced volume of water was determined. The density of the water used was also noted. Using the weight of displaced water collected in the centrifuge tube and the density of water, the volume of displaced water was determined.

 

3. RESULTS AND DISCUSSION:

The developed apparatus is shown in Figure 1.

 

Figure 1: Developed apparatus for determination of paw volume

 

Calibration of the apparatus:

To the pre-filled apparatus, different volumes of water (0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 mL) were added. Each time the displaced quantity of water was collected into a pre-weighed microcentrifuge tube.  The volume of displaced water was determined (Table 1). The experiment was carried out in triplicate. Figure 2 shows the calibration plot obtained with the developed apparatus. The obtained plot was linear with a high R2 value of 0.983. Thus it was confirmed that the apparatus is useful for the determination of volume.

 

Table 1:  Data of calibration plot

Volume of water added (mL)

Volume of determined by the developed method ± SD (mL)

0.05

0.180±0.047

0.1

0.208±0.035

0.2

0.253±0.045

0.3

0.337±0.020

0.4

0.447±0.105

0.5

0.587±0.007

0.6

0.682±0.073

0.7

0.671±0.048

0.8

0.776±0.067

0.9

0.872±0.078

1.0

1.033±0.045

 

Figure 2: Calibration plot of water displacement for the developed apparatus

 

4. SUMMARY AND CONCLUSIONS:

A new apparatus was developed for the determination of paw volume of rats. The calibration plot obtained with the developed apparatus was linear with a high R2 value of 0.983. Thus it was confirmed that the apparatus is useful for the determination of rat paw volume.

 

5. CONFLICT OF INTEREST:

USJ has made a patent application (Indian Patent Application No. 201941009319 dated 11th March 2019) related to this work.

 

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Received on 19.06.2019            Modified on 28.07.2019

Accepted on 13.09.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(5):2373-2375.

DOI: 10.5958/0974-360X.2020.00426.6