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1. Divide rats into groups as required for experiment and label appropriately (e.g., using cage cards, ear tags, or ear notches; see unit 1.5). Label one group as control rats (to be injected with saline) and another group as experimental (to be injected with 10S PG-PS).

2. Weigh rats and determine average body weight of each group.

3. If arthritis severity is to be monitored by measuring ankle diameter (also see step 8), individually measure maximal lateral ankle diameter of each rat with a caliper as a baseline measurement. Take measurements three times for both ankles and calculate average diameter .

Also see unit 15.5. Alternatively, arthritis may be assessed by clinical observation (see step 8).

4. If 10S PG-PS suspension has been stored >2 days or frozen: Sonicate 10S PG-PS suspension 10 sec at low energy setting, room temperature.

If the commercial preparation of 10S PG-PS is used (supplied as a white opalescent liquid suspension), it should be vortexed 30 sec or sonicated 10 sec at low energy setting prior to injection.

5. Calculate volume of 10S PG-PS suspension to inject on the basis of 10 to 60 ^g rhamnose per g average body weight of rats.

Support Protocol 2 describes measurement of PG-PS concentration in terms of rhamnose concentration. Preliminary studies should be done to assess the relative potency of each 10S PG-PS preparation because preparations may vary. The higher the dose of 10S PG-PS, the higher the incidence and severity of arthritis; however, higher 10S PG-PS doses also result in higher mortality. See Critical Parameters for discussion of dosage considerations.

6. Using a 1-ml syringe and a 23-G needle, inject each test rat with the amount of 10S PG-PS calculated in step 5, intraperitoneally (unit 1.6) into the left lower abdomen to minimize the possibility of injecting the cell wall fragments into the stomach or cecum.

If the rat is properly restrained, anesthesia is not necessary.

7. Inject each control rat as described in step 6, but substitute sterile 0.85% saline for the 10S PG-PS.

8. Depending on the experimental design and objectives, evaluate each animal daily for arthritis for the first 6 days after injection, then every 2 to 3 days thereafter for 6 to 8 weeks, using either of the following methods.

a. By measuring ankle diameter: Measure each ankle three times with calipers as described in step 3 (also see unit 15.5). Average the three measurements and record as a single data point. Plot data over time to present the progression of arthritis graphically.

b. By clinical observation: Grade extremities distal to the elbow or knee on a scale of 0 to 4, based on the number of joints involved, the degree of erythema (redness) and swelling, and the degree of distortion of normal joint contours (also see unit 15.4, Basic Protocol and Commentary). Plot data over time to present the progression of arthritis graphically.

A score of 0 signifies normal (no arthritis); 1 signifies barely perceptible arthritis; 2 signifies definite but mild arthritis; 3 signifies severe arthritis; and 4 signifies extremely severe arthritis (see unit 15.4 for more detail). The scores for each joint are summed; thus, the maximum score for an individual rat is 16. The scores for all animals in a group are averaged to generate a single data point. The advantage of the clinical scoring method is that is can be done frequently, reproducibly, and rapidly. With a short period of training, a single observer can reproducibly assess arthritis severity.

It may also be desirable to monitor body weight (Fig 15.4.2) or other experimental variables. Typically, most relevant experimental data can be obtained over a 6- to 8-week period of observation.

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