Microprocessor-assisted plethysmograph for the measurment of mouse paw volume

Microprocessor-assisted Plethysmograph Measurment of Mouse Paw Volume

EDWARD F. WEBB AND DON

for the

E. GRISWOLD

A sensitive and reproducible method of measuring mouse paw volume was developed by interfacing a Mettler DeltaRange top-loading balance with a microcomputer. This methodology combined ease of operation and precision with the advantages of computer-controlled data processing and archivable storage of data. Key Words:

Plethysmograph;

Quantitation

of inflammation;

Edema

INTRODUCTION Plethysmography is a widely applied principle in the measurement of rat and mouse inflammatory responses (Griswold et al., 1982a; Griswold and Walz, 1982b; Holsapple et al., 1980; Loubaris et al., 1983). Its usefulness has been extended to the measurement of contact sensitivity (Griswold and Walz, 1982b) and evaluation of tumor growth (Laing et al., 1973). Several methods have been utilized, including measurement of mercury displacement using a pressure transducer and galvanometer-coupled apparatus (Winter and Risley, 1962). This methodology represented an improvement over earlier methods that used direct measurement of displaced fluid (Buttle and D’Arcy, 1957). More recently, an improved method using a Mettler PI210 balance to quantitate the pressure required to insert a rodent limb into a mercury bath has been described (Hall and Hallett, 1975). This method was designed to be used with rats and other large rodents. A modification of this method, designed primarily for use with mice, is described in this report. This method combines sensitivity and ease of reading with automatic electronic data collection and processing. METHOD Plethysmograph

Description

A plexiglass table with a hole for the mercury reservoir to pass through is used to provide support for the person making the volume measurements. The table was placed over a Mettler PC440 DeltaRange top-loading balance. The mercury reservoir (20-ml screw-cap vial containing 19 ml of Hg) was placed on the balance. Fromthe Department of Immunology, Smith Kline & French Laboratories, Philadelphia, Pennsylvania Address reprint requests to Dr. D. E. Griswold, Department of Immunology, Smith Kline and French Laboratories, 1500 Spring Garden Street, Philadelphia, PA 19101. Received February 1,1984; revised and accepted May Ii’, 1984.

149 Journal

of Pharmacological

0 1984 Elsevier

Science

Methods Publishing

12, 149-153 Co.,

Inc.,

(1984)

52 Vanderbilt

0160~54021&l/$03.00 Avenue,

New

York,

NY 10017

150

E. F. Webb and D. E. Griswold

The Mettler CL249 interface connected to the balance’s 03 serial data port was connected to the SADI, an IEEE 488-RS-232 interface (Connecticut Microcomputer, Inc.), which was in turn connected to the IEEE 488 port of a Commodore PET 4032 microcomputer. A foot switch connected to the user port of the microcomputer enables the operator to send the readout in grams directly from the balance at the precise moment desired. Software that monitors the foot switch and balance, calculates the regression line, and calculates the paw volumes was written in our laboratory. Cylinders of known volume were used to calibrate the instrument. The paw displacement in grams was then converted to volume and printed on paper and/or magnetic tape for later data analysis. A schematic diagram of the paw volume measurement and processing can be seen in Figure 1. Plethysmograph

Calibration

Eight Teflon cylinders of known volume (83, 154, 158, 161, 172, 178, 228, and 250 PI) were used to generate the calibration curve (regression line). Each cylinder was immersed into the mercury, and the displacement in grams measured by the balance was collected by the computer after the foot switch was depressed. After all eight cylinders were measured, the computer software calculated the regression line for paw volume calculation. Measurement

of Mouse Paw Volume

The tibia1 tarsal joint of each hind limb of the mouse was marked with a black, water-resistant marking pen, and each limb was immersed in the mercury to this mark. Each limb was extended so that it was perpendicular to the mercury for an accurate and consistent reading.

Sadi Serial l/O Interface IEEE 488-RS-232

Telephone Modem To Mainframe Computer

Mettler Interface CL-249-RS-232

1

Commodore Pet 4032

PI

Mettler PC440 Balance With 03 Data Port

FIGURE 1.

P

Printer

Cassette Data Storage

. _... Switch

Diagrammatic

representation

of paw volume measuring apparatus.

Computer-Assisted

I

Mouse Plethysmograph

I

I

I

50

150

250

Volume(PI) FIGURE 2. Scatter plot and regression line for standard volumes used to calibrate instruand 250 ~1. ment. Standard volumes = 83, 154, 164,172,178,228,

Histamine

Edema and Plethysmographic

Measurement

Male Balb/c mice, 22-25 g (Charles River Breeding Labs) were injected S.C. in the right hind paw with histamine at concentrations of 0.18, 1.8 and 18 kg in IO ~1 of saline. The control group received 10 ~1 saline in the right hind paw. Edema was assessed 10 min later by measuring the treated and untreated paw and subtracting untreated from treated to get the increase in volume. Chlorpheniramine (5 mg/kg i.v.) was given immediately before histamine injection. RESULTS AND DISCUSSION A scatter plot and regression line of known volumes in microliters versus weight of mercury displaced in grams can be seen in Figure 2. Each point represents a mean of three measurements. A relationship between volume and weight was in-

TABLE 1 STANDARD

Operator VOLUMES

Accuracy: Actual versus Measured Volumes of Test Cylinders MEASURED

VOLUME

DIFFERENCE

(w)

(J*L)

(GIL)

154 158 164 172 178 228 250

148 162 168 177 183 228 242

-6 +4 +4 f5 +5 0 -8

PERCENT

a Teflon cylinders measured by calipers and volume calculated using formula V = ll Ph.

DIFFERENCE

4 3 2 3 3 0 3

151

152

E. F. Webb and D. E. Griswold

I

_

0.18 Histamine

Hlstamme + Chlorphemramme

,

I

1.8

18

Concentration

(pg)

FIGURE 3. Histamine dose-response curve with chlorpheniramine as competitive antagonist. Chlorpheniramine was given i.v. at the same time as the histamine injection in the paw. Paw volumes measured 10 min post-histamine injection. Means +S.D.; n = 5.

dicated by a correlation coefficient of 0.994. The line has a slope of 13.50: 1, which reflects both the sensitivity and precision with respect to the specific gravity of mercury (13.60 at 0°C). Table 1 demonstrates the accuracy of measurements made by a single operator. Cylinders of known volume were each measured by the calibrated plethysmograph, and the difference between the actual and the plethysmographically measured value was determined. The range of the percent difference was O-4%, which can be attributed primarily to operator error. An example of results from a paw edema study using histamine and a competitive antagonist, chlorpheniramine, obtained with this balance method can be seen in Figure 3. Three concentrations of histamine, 0.18, 1.8 and 18 t.r,g,were injected into the right hind paw of mice treated immediately before with either saline or chlorpheniramine (5 mg/kg). A linear dose response was seen with histamine. The apparatus proved to be sufficiently sensitive to detect a significant antagonism by chlorpheniramine. This methodology combines ease of operation with sensitivity and reproducibility+ This has been achieved by using a Mettler DeltaRange top-loading balance coupled to a microprocessor. Additional advantages of this apparatus include computercontrolled data processing, formatting, and archivable storage of data. The ease and rapidity with which unanesthetized animals may be measured also facilitates the evaluation of time-course experiments. We would writing

the

preparation

like to express software of this

for

this

manuscript.

our

thanks

to Mr.

plethysmographic

Julius

Schainbaum

method,

for

and to Ms.

his assistance Jane Manze

for

and guidance her

help

in

in the

Computer-Assisted

Mouse Plethysmograph

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