INTRODUCTION

     Respiration is regulated by the pCO₂, pO₂, and pH of the plasma and cerebrospinal fluid (csf).  The levels of these factors are sensed by the chemoreceptors located in the carotid sinus, the aortic arch, and the medullary respiratory centers.  Afferent sensory nerves run from the peripheral chemoreceptors to the medulla via the spinal column.  The sensory fibers from the aortic arch are contained in the vagus nerves.

     The lung stretch receptors, which sense the rate and amount of the lung inflation, also run in the vagi.  These fibers conduct an increased frequency of action potentials when the lungs are inflated and subsequently shut off the inspiratory center neurons.  Exhalation, primarily a passive process, then occurs.  In the previous set of laboratories, you already noticed that respiration temporarily stop during the stimulation of an intact or the cephalic end of a cut vagus nerve.  This was due to the stimulation of these sensory fibers originating in the stretch receptors in the lungs.  Please review these concepts in your lecture notes and assigned readings in the text.

     Dissolved oxygen (pO₂ in mm Hg) represents only a small fraction of the blood's O₂ content.  Under normal conditions, pO₂ reflects the total O₂ carried by the blood (a combination of dissolved and hemoglobin transported O₂).  Under what conditions is this relationship altered?  Consider anemia and CO (carbon monoxide) poisoning.

     The  plasma pCO₂ is directly related to the plasma pH as illustrated by the following familiar reaction that is catalyzed by the enzyme carbonic anhydrase:

Thus, as plasma CO₂ increases due to a decrease in alveolar ventilation, the plasma pH decreases.  More importantly, the increased plasma CO₂ diffuses into the csf and is then converted into H⁺ and HCO₃^- .  As the csf pH falls, the medullary chemoreceptors are stimulated and the rate and depth of respiration should increase.

EXPERIMENTAL DESIGN

     In this set of experiments, you will alter the O₂ and CO₂ content of the inspired air and note any changes in the rate and depth of respiration and in the HR & BP.  You will also introduce a 'fixed' metabolic acid and possibly an inhibitor of carbonic anhydrase into the plasma, and record any resulting changes in these parameters. Of course you will be expected to explain the results based upon the theory discussed above.

EXPERIMENTAL PROCEDURES

Prepare an animal for recording BP and respiration as described in the previous exercise.  A respirator may be used only in times of distress, i.e., to save the animal if BP falls and breathing fails.

The following set of treatments will be given:

1.     Asphyxiation:
Place your finger firmly over the tracheal tube for 30-45 sec. Remember, do not asphyxiate unti lthe animal stops trying to breathe!!!! 30-45 sec should be a sufficient amount of time for a buildup of CO₂!

2.     Open dead space:
 Fit an additional piece of tubing or Pasteur pipette snugly into the tracheal cannula.

3.     Rebreathe:
 Fit a short piece of tubing sealed with the finger of a latex glove snugly into the  tracheal cannula.
 
4.     Rebreathe with soda lime:
 Repeat the rebreathe experiment, but include a small amount of soda lime in the  finger of the latex glove.

5.     Lactic acid:
Inject incrementally larger doses of lactic into the jugular cannula. You should use the same volume of acid for each injection so you will need to make a number of dilutions of the stock solution prior to the experiment. Use caution
in this step as the rats react quickly. Wait at least three minutes between doses and be sure that the animal has sufficiently recovered before administration of the next dose.

6.     Increased CO₂ levels:
With the assistance of your TA, connect the tracheal cannula to tubing attached to the cylinder containing CO₂ for incrementally long periods of time (Time it!!). Remove the CO₂ source and observe rat's reaction.

7.      Acetazolimide (carbonic anhydrase inhibitor):
Inject 0.2 ml of acetazolimide at a concentration of 10 mg/ml (be sure that solution is well mixed before injection). Note that acetazolimide is not soluble in saline, but is dissolved in a DMSO solution. What additional experiment must you perform in order to take this into account?
Repeat any of the above experiments after the acetazolimide injection.