(Calvero, 2006)
INHIBITION OF
OXYTOCIN INDUCED MYOMETRIAL CONTRACTIONS USING IBUPROFEN
Katherine A. Matthews
Supervisor: Dr. Brenda Peters
5-7-2007
Key Words:
Pre-term labor
Oxytocin in Parturition
PGF2a in Parturition
Ibuprofen in Parturition
Inhibition of uterine contractions
COX-2 Inhibition
ABSTRACT
The magnitude of the role PGF2a has on labor contractions is not completely known. There is evidence to suggest an intricate association between oxytocin and prostaglandins in the production of mammalian myometrial contractions. The binding of oxytocin to cell membrane receptors initiates a series of secondary messengers within the cell. COX-2, an enzyme associated with this secondary messenger pathway, produces a common prostaglandin precursor. Ibuprofen works by inhibiting the COX-2 enzyme. Through inhibition, prostaglandins will not be produced, and myometrial contraction should be inhibited. This hypothesis was tested using pregnant rats in late gestation. Results indicated that at high ibuprofen concentrations, myometrial contractions were significantly inhibited. From these findings, we believe that prostaglandins do play a significant role in myometrial contractions.
INTRODUCTION
Oxytocin, derived from the Greek words oxus, meaning sharp, and tokos, meaning childbirth, is a peptide hormone consisting of nine amino acids (Mitchell et al, 1998, Engstrom, 2002). The amino acids are: NH2 – Gly – Leu – Pro – Cys – Asn – Gln – Ile – Tyr – Cys, with a disulfide linkage between the two Cys residues (Messer, 2000).
Figure 1 – Structure of Oxytocin
(Calvero, 2006)
This hormone, to date, is the most potent stimulant of uterine contractions in mammals, and is administered in the clinical setting to induce labor (Serradeil-Le Gal et al, 2004).
Oxytocin is produced in the paraventricular and supraoptical nuclei of the hypothalamus, and stored in the posterior pituitary gland (Engstrom, 2002). Cervical distension within the uterus causes an action potential to travel to the hypothalamus, signaling the production and release of oxytocin (Blanks et al, 2003). During pregnancy, the quantity of oxytocin produced by the hypothalamus increases by roughly 50% (Russell et al, 1998, Blanks et al, 2003). Once in circulation, it travels through the blood to its target site. Oxytocin has a half-life of 5-15 minutes in circulation, after which it is degraded in the liver by oxytocinase (Engstrom, 2002). In addition, oxytocin is synthesized locally within the epithelium of the uterus (endometrium) (Blanks et al, 2003). Local and hypothalamic oxytocin leads to myometrial contractions, which ultimately leads to the expulsion of the young (parturition) (Russell et al, 1998, Blanks et al, 2003).
Oxytocin directly and indirectly initiates contractions when it binds to its receptor, located in the myometrium. The oxytocin receptor is a seven transmembrane domain receptor, containing 389 amino acid residues (Engstrom, 2002, Mitchell et al, 1998). This receptor is coupled to a G protein. The binding of oxytocin to its receptor activates the G protein, and the a and bg subunits of the G protein dissociate. The bg subunit then activates phospholipase C, which converts phosphatidyl inositides to diacylglycerol and inositol-1,4,5-triphosphate (IP3). The direct activation of contraction involves IP3. IP3 stimulates the release of calcium (Ca2+) from the cells sarcoplasmic reticulum and additionally increases the flux of Ca2+ into the muscle cell from extracellular sources. Ca2+ then binds to calmodulin, stimulating myosin light chain kinase activity, which leads to smooth muscle contraction
Figure 2 – Mechanisms of Action of Oxytocin
(Mitchell et al, 1998).
Indirect initiation of contractions is the result of diacylglycerol, which is involved in the synthesis of prostaglandin F2a (PGF2a) (Engstrom, 2002, Mitchell et al, 1998, Gimpl et al, 2001, Carsten, 1973).
Prostaglandins play an integral role in many reproductive events, especially luteolysis and parturition (Parent et al, 2003). Prostaglandins are 20-carbon chain fatty acids that function as paracrine hormones (Olson, 2003, Engstrom, 2002 ).
Figure 3 – Prostaglandin F2a
(Chiabrando et al, 2002)
15-hydroxyprostaglandin dehydrogenase (PGDH) is the principle enzyme that degrades prostaglandins. This enzyme can be found in high concentrations in the lungs, liver and kidneys, which explains why prostaglandins function as local, rather than endocrine, hormones (Engstrom, 2002, Thorburn et al, 1979). This enzyme is under the control of progesterone and estrogen, which will be discussed later in this paper.
The prostaglandin that plays the greatest role in the degradation of the corpus luteum (luteolysis) and parturition is PGF2a, which is produced in the endometrium. PGF2a is a product of the oxytocin biological pathway. The oxytocin biological pathway initiates the synthesis of PGF2a through diacylglycerol. Diacylglycerol activates protein kinase C, which acts to liberate arachidonic acid from phospholipids in the cell plasma membrane. Arachidonic acid is a polyunsaturated fatty acid, which acts as the common precursor to all prostaglandins.
Figure 4 – Arachidonic Acid
(Dharmananda, 2003)
Arachidonic acid undergoes a series of oxidation-reduction reactions by the enzyme cyclooxygenase-2 (COX-2), which forms the unstable endoperoxide, PGH2. PGH2 is the common prostaglandin precursor. Finally, a cell specific isomerase catalyzes the reaction of PGH2 to the cell specific prostaglandin (in our case, PGF2a) (Olson, 2003, Parent et al, 2003, Okawa et al, 2001, Strakova et al, 1998, Engstrom, 2002, Nathanielsz, 1978, Mitchell et al, 1998, Thorburn et al, 1979, Chan et al, 1982, Russell et al, 1998).
Figure 5 – Synthesis of PGF2a
PGF2a is then exported from the cell and binds to FP receptors in the myometrium, which are specific for the prostaglandin F2a isoform. This receptor is a seven transmembrane domain receptor with 366 amino acid residues (Engstrom, 2002). The FP receptor is coupled to a G protein, like the oxytocin receptor, and mobilizes intracellular and extracellular Ca2+ in the same manner, leading to contraction of that cell. The FP receptor also activates phospholipase C, producing diacylglycerol. Diacylglycerol ultimately leads to the production of more PGF2a. Thus, PGF2a works directly on the myometrium to induce contraction, and indirectly to produce more PGF2a (Engstrom, 2002, Olson, 2003, Celik et al, 2002, Phillippe et al, 1997).
The above mechanisms of action are proposed to explain the events that lead to parturition in mammals. During pregnancy, however, these mechanisms are shut down and the myometrium is unresponsive to the actions of oxytocin (Russell et al, 1998). This is due to the low density of oxytocin receptors present in the endometrium of the pregnant uterus. Regulation of these receptors is influenced by the ratio of progesterone and estrogen systemically (Nathanielsz, 1978).
The corpus luteum is present during gestation in the rat, and produces large quantities of progesterone (Nathanielsz, 1978). High progesterone levels cause saturation of progesterone receptors in the endometrium, inhibiting the synthesis of local endometrial oxytocin receptors. Also, progesterone activates PGDH, which degrades any prostaglandin present in the pregnant uterus (Thorburn et al, 1979). In this way, the pregnant uterus is unresponsive to oxytocin, thus inhibiting the production of PGF2a.
In the days leading to parturition, oxytocin synthesis in the endometrium increases. This causes PGF2a to accumulate in the uterus. PGF2a induces luteolysis, changing the progesterone-estrogen balance in mammals to favor estrogen. The result is a dramatic decrease in systemic progesterone levels (Gimpl et al, 2001, Parent et al, 2003). With progesterone no longer blocking the synthesis of oxytocin receptors, up regulation occurs in the endometrium and myometrium and the uterus becomes responsive to oxytocin (Strakova et al, 1998). Also, PGDH activity is inhibited by estrogen, and the concentration of PGF2a accumulates in the uterus (Thorburn et al, 1979).
There are a number of pharmaceutical drugs that can inhibit prostaglandin production. These drugs are categorized as NSAIDs (Non-steroidal Anti-inflammatory Drugs). Ibuprofen is one such drug.
Figure 6 - Structure of Ibuprofen
(The chemical structures for common named molecules, 2004)
It was approved by the FDA in the 1970’s and is one of the newest NSAIDs on the market. NSAIDs, referred to as prostaglandin synthetase inhibitors, directly block the production or prostaglandins. Ibuprofen acts as a reversible inhibitor to the COX-2 enzyme. The COX-2 isoform is directly associated with PGF2a synthesis and is predominately located in the uterus. This enzyme catalyzes the direct conversion of arachidonic acid to the common prostaglandin precursor PGH2. Blocking this enzyme results in the inhibition of prostaglandin production (Chan, 1983, Chan et al, 1982).
To date, the biological processes behind parturition in mammals are not completely understood. A relationship has been established between oxytocin and PGF2a, but the magnitude of this relationship on parturition is not definite. By inhibiting the production of PGF2a using ibuprofen, the role that prostaglandins play in parturition can be analyzed. My research hypothesis is that prostaglandins play a significant role in the mechanism of action of oxytocin on myometrial contractions in rats. By blocking prostaglandin synthesis using ibuprofen, we anticipate a dramatic decrease in the oxytocin induced contractility of the rat myometrium. A finding contrary to this would implicate that PGF2a does not have as significant a role as previously thought.
MATERIALS AND METHODS
The procedures and method of euthanasia was approved by the St. Ambrose University ad-hoc Animal Care and Use Committee. Information regarding the established committee and its members can be found in Appendix B.
MATERIALS
Six female Sprague-Dawley rats were used, weighing between 300-375 grams. Two were non-pregnant and four were pregnant, on their 18th day of gestation (out of 21 days). All rats had normal oestrous cycles. The rats were purchased from Zivic Labs, Inc. Rat food, bedding, water bottles and food dishes were purchased from Petco. Handling gloves were donated by Kathy Van Buer, D.V.M. Rat cages were previously purchased by the SAU Biology Department. A guillotine (NEMI model 701) was purchased from NEMI Scientific.
The tissue bath apparatus used was purchased from CB Sciences, Inc. (iworx STB-125, Student Tissue Bath). This apparatus included: 1 steel rod, 1 plastic base, 1 force transducer, 3 stand clamps, 1-25 mL tissue chamber, 1 glass tissue support rod, nylon tubing, 1 oxygen connector, silicone tubing, 1 pinch clamp and tissue clips. Silk thread was purchased from Quilts by the Oz. The thermocycler water bath used was property of the St. Ambrose University Chemistry Department. The O2-CO2 gas canister used to bubble our Ringer’s solution within the tissue bath was purchased from Scott Specialty Gases.
District Drugs, in Rock Island, Illinois, supplied the oxytocin and ibuprofen-sesame seed oil solution. PGF2a was purchased from Sigma-Aldrich Co. The prostaglandin solution was stored in a light resistant bottle to prevent degradation. All constituents of Ringer’s Solution were obtained through the St. Ambrose University Biology and Chemistry Departments. Syringes were donated by Jim Perry of District Drugs.
The computer program used for data acquisition and analysis was Labscribe. The data acquisition unit was iworx AHK/214, which had been previously purchased by the St. Ambrose University Biology Department from CB Sciences, Inc. Minitab Release 14 Statistical Software was used to analyze the data; it was donated by Timothy Matthews.
METHODOLOGY
For our controls, sesame seed oil was heated to body temperature in a warm water bath. 0.20mL was drawn into a 21 gauge syringe. For our experimental groups, 0.20 – 0.60mL of a 50mg/mL ibuprofen-sesame seed oil solution (Powell et al, 1982) was heated in a warm water bath to body temperature and drawn into a 21 gauge syringe. For both our controls and our experimental groups, the injections were administered intramuscularly in the morning hours of 8-9 am into the right flanks of the rats. Two hours after injection, the rats were euthanized by decapitation. A small abdominal incision was then made and the uterine horns were located and excised. Incisions were made at the lateral positions of the horns, and each rat pup was removed from the horn. The pups were then euthanized by cervical dislocation. The horns were then placed into a water bath in a 500 mL beaker containing Ringer's solution at 37 C. 15 cm long sutures were threaded through the anterior and posterior ends of each uterine horn. Each horn was used within two hours of excision. The apparatus described in Appendix A was set-up prior to euthanization. Also, 30 mL of Ringer’s solution was added to the tissue bath chamber. The water bath maintained this solution at 37 C in the experimental apparatus (Celik et al, 2002, iworx physiology laboratory manual, Labscribe).
One end of the uterine horn was tied to the glass tissue support rod and lowered into the tissue bath chamber. The suture at the other end of the horn was held out of solution and attached to the hook on the force transducer (* Note – we made sure the uterine horn was not in contact with the walls of the tissue bath chamber). The nylon tubing was then connected to the O2-CO2 gas canister and the Ringer’s Solution in the tissue bath chamber was gently bubbled (Celik et al, 2002, iworx physiology laboratory manual, Labscribe).
At this point, the iworx- AHK/214 data acquisition unit was turned on; the Labscribe program was set to record smooth muscle contractions, and normal uterine muscle activity was recorded for ten minutes (Celik et al, 2002, iworx physiology laboratory manual, Labscribe).
In CONTROL SET I, different concentrations of oxytocin were tested to determine the concentration that led to optimal uterine muscle contraction. The following concentrations were tested: 0.05 U/mL, 0.1 U/mL, and 0.5 U/mL (Okawa et al, 2001, Chan et al, 1982).
In CONTROL SET II, uterine contractility in the presence of different concentrations of PGF2a was measured. The following concentrations were used: 1nM, 10nM, 100nM, 500nM, and 1mM (Celik et al, 2002, Phillippe et al, 1997).
In the EXPERIMENTAL GROUPS, the effect of ibuprofen on uterine muscle contractions in the presence of oxytocin was investigated. 10 mg, 30 mg and 40 mg of ibuprofen were administered (Powell et al, 1982). The concentration of oxytocin used was 0.1 U/mL.
To obtain data from our controls and experimental groups, the iworx AHK/214 Labscribe program began recording muscle activity for ten minutes. We then added the desired amount of oxytocin or PGF2a to the Ringer’s solution in the tissue bath chamber and recorded activity for another ten minutes. At this point recording halted. The uterine horn was then carefully removed from the chamber, the Ringer’s solution was emptied from the chamber, and the chamber was cleaned out. Preparations were made for the next experiment or the lab was cleaned up for the day (Celik et al, 2002, iworx physiology laboratory manual, Labscribe).
The data from the different control and experimental groups was then analyzed based on contraction frequency and amplitude as recorded by the iworx AHK/214 data acquisition system. Amplitude was measured from baseline to the peak of muscle contraction. Frequency of muscle contractions was measured as the inverse of the time from one peak to the next (Celik et al, 2002, iworx physiology laboratory manual, Labscribe).
Data obtained was statistically analyzed using ANOVA analysis. ANOVA stands for analysis of variance. This computer system found the mean and standard deviation of the input data and tested the null hypothesis, stating that there is no significant difference between two groups. From the two groups mean and standard deviations, the data’s z score was found. The probability correlated with that z score was used to find the P value. The P test was based on a 95% confidence interval. A P value of 0.05 or less was deemed as statistically significant with the above parameters in place. In this way, the computerized P value analyzed the statistical significance of the data.
RESULTS
In all, six rats were used in this project ( two non-pregnant and four pregnant). Sixteen trials were conducted, and the data obtained was analyzed statistically using ANOVA analysis.
PREGNANT vs. NON-PREGNANT RATS
The first four trials were conducted using two non-pregnant rats’ uterine horns. In the first two trials, the horns were suspended in a 0.05 U/mL oxytocin solution and myometrial activity was recorded - no change in contractility was observed. In trials three and four, the horns were suspended in a 0.5 U/mL oxytocin solution. Once again, no observable change in contractility was observed. The next twelve trials were conducted with pregnant rats. In trial six, pregnant horns were suspended in a 0.5 U/mL oxytocin solution. In a 0.5 U/mL oxytocin solution, non-pregnant and pregnant uterine horns were compared. The non-pregnant uterine horn's average amplitude was 0.17190 +/- 0.02554, while pregnant rat horn's average amplitude was 0.22588 +/- 0.10475 Observing Figures 6 and 7, the pregnant horns were significantly more contractile than non-pregnant horns (P = 0.033).
No significant difference in frequency of contraction was observed, however, between pregnant and non-pregnant horns.
CONTROLS – OXYTOCIN
Trials five through seven served as our controls. Trial five was in the presence of a 0.1 U/mL oxytocin solution and trials six and seven were in the presence of 0.5 U/mL oxytocin solutions. Due to difficulties with the experimental apparatus, data from trial seven was unusable. However, in all control trials, the general trend observed was that the horn's amplitude of contraction increased in the presence of oxytocin. In trial five, the mean amplitude of contraction before the addition of oxytocin was 0.0503 +/- 0.0175, while the mean amplitude after the addition of oxytocin rose to 1.0405 +/- 0.1578. These results indicated that horns were significantly more contractile in the presence of oxytocin (P = 0.000). In trial six, the mean amplitude of contraction before the addition of oxytocin was 0.12991 +/- 0.08515, while the mean amplitude after addition rose to 0.22588 +/- 0.10475. These results also indicated that horns were significantly more contractile in the presence of oxytocin than in its absence (P = 0.016). Referencing Figures 8-10, however, indicates that the amplitude of contraction more dramatically increases in the 0.1 U/mL oxytocin solution than in the 0.5 U/mL solution. From these results, experimental oxytocin concentrations were held constant at 0.1 U/mL of oxytocin.
No significant change in the frequency of contraction was detected at either concentration of oxytocin.
EXPERIMENTAL GROUPS – IBUPROFEN
Trials eight through sixteen were conducted using three pregnant rats. Trial eight through ten investigated the effects of 10 mg of ibuprofen; trials eleven through thirteen investigated 30 mg of ibuprofen and trials fourteen through sixteen investigated 40 mg of ibuprofen. Referencing Figures 11 and 12, in the presence of 10 mg of ibuprofen, the horn's contractility significantly increased after the addition of oxytocin (P = 0.000). Figures 11 and 13 show that in the presence of 40 mg of ibuprofen, however, the addition of oxytocin did not significantly affect the contractility of the horn (P = 0.521). In the presence of 30 mg of ibuprofen, the contractility of the horns was shown to significantly decrease after the addition of oxytocin (P = 0.003). Trial twelve could not be analyzed due to difficulties with the experimental apparatus, which flawed the data.
No significant change in the frequency of contraction was detected during any of the experimental trials.
DISCUSSION
Pregnant vs. Non-pregnant Rats
Oxytocin receptors are regulated due to the antagonistic effects of estrogen and progesterone. At high estrogen levels, oxytocin receptors are up regulated; at high progesterone levels, oxytocin receptors are down regulated. In the pregnant rat, progesterone levels are systemically high until the end of gestation; on day 18 of gestation (out of 21 days), the balance between estrogen and progesterone shifts, favoring estrogen. Oxytocin receptors are then up regulated in the rat endometrium and the pregnant uterus becomes responsive to the actions of oxytocin (Nathanielsz, 1978).
While researching this experiment, there was conflicting data pertaining to the ability of non-pregnant rats to respond to oxytocin. Some data stated that non-pregnant uterine would be induced to contract using oxytocin (Thorburn & Challis, 1979), while others suggested it could not (Chan et al, 1982). As our results indicate, the non-pregnant horns were not responsive to the actions of oxytocin. We surmise this occurrence was due to the low density of oxytocin receptors present on the non-pregnant horns. Without microscopic or chemical analysis, we can not definitively conclude this, but we believe the non-pregnant rats used had either not yet reached sexual maturity or were at a stage in their reproductive cycles when progesterone concentrations were high. The resulting factor was that oxytocin receptor up regulation had not occurred, which rendered the non-pregnant horns unsusceptible to the actions of oxytocin.
The results from the pregnant rats indicated their horns were susceptible to oxytocin. We used pregnant rats on their eighteenth day of gestation for the remainder of the experiment. Since the pregnant rats’ balance between progesterone and estrogen shifts to favor estrogen around day eighteen, oxytocin receptors are then up regulated and the uterus becomes susceptible to oxytocin (Nathanielsz, 1978).
Controls with Oxytocin
To determine our experimental concentration of oxytocin, we analyzed the pregnant horn's contractility in response to several different concentrations of oxytocin. The most prominent contractions observed were in the presence of 0.1 U/mL of oxytocin. From our trials, we believe this to be the optimal concentration to induce contractions. Concentrations higher than this amount did not cause the same magnitude of contraction. Initially, this result was surprising, especially under the assumption that increasing the oxytocin concentration should increase the magnitude of contraction. It may be true, however, that too much oxytocin can stimulate too many receptors, resulting in a generated force of contraction that is great enough to damage the muscle. Causing damage to the muscle could decrease the muscles contractility, which may have resulted in the decreased amplitudes we observed during these control trials.
As another control, we intended to test the ability of PGF2a to induce contractions on the uterine horns. As a result of time and financial constraints, we were unable to perform these trials. We felt it was most pertinent to test oxytocin and establish our experimental concentrations. This is a flaw within our experiment, however, and if we were to repeat this research project, we would examine PGF2a’s effect on myometrial contractions.
IBUPROFEN
Our data revealed that high dosages of ibuprofen are needed to significantly inhibit oxytocin induced contractions. At 10 mg, ibuprofen had little to no effect on decreasing the contractility of the horns. However, at 40 mg, ibuprofen was shown to significantly inhibit oxytocin induced contractions. As the data shows, the mean amplitude of contraction before and after the addition of oxytocin remained nearly unchanged. In the case of our 30 mg trials, however, the data showed a significant decrease in muscle contractility after the addition of oxytocin to the horns. This is by no means representative of the inhibitory effects of ibuprofen on oxytocin induced myometrial contractions. Inhibition by ibuprofen should show little to no change in the amplitude of contraction before and after the addition of oxytocin. There is no biological or chemical explanation we can provide for this finding. We had several difficulties during this trial, including inaccurate injections and increased handling, which could have altered our results. We believe the results from these trials are not representative of ibuprofen and are the result of experimental error.
We would like to point out, however, that the dosage of ibuprofen we used in this research project to inhibit contractions is much higher than standard dosages administered in the clinical setting. The standard dosage of ibuprofen is 16 mg/kg of rat weight (Drugs used in inflammatory conditions and their effects on wound healing, 2005). The dosage that inhibited oxytocin induced contractions in this project was about 130 mg/kg of rat weight. We had approval from the veterinarian on our Animal Care and Use Committee to inject this dosage. Toxicology studies would be needed to determine the true effect of this dosage on the rat’s body. No toxicity effects are apparent on muscle tissue at high dosages, however, which provides evidence that inhibition of contraction was due to inhibiting the COX-2 enzyme and not due to degradation of the muscle tissue (Totten et al, 2004).
There are other concerns involving ibuprofen as well. Literature cites that ibuprofen could be detrimental to the growing fetus (Russell & Leng, 1998). Further research on this topic needs to be considered if ibuprofen is looked at further as a possible treatment for pre-term labor.
Frequency
Through out this experiment, data on the frequency of contraction was inconclusive. Our data indicated that one trial's frequency of contraction decreased, while the next increased in the presence of oxytocin. There is no way to make any assertions about this data. At this point, we can give no explanation for our findings.
Conclusion
Since this was our first experience with this research project, experimental errors can account for some of the discrepancies observed. Changing of the experimental method was common through out the project to account for new problems that were unforeseen when preparing for the trials. All in all, we did provide evidence for several factors. First, we showed that pregnant horns are more susceptible to the actions of oxytocin than non-pregnant horns. Secondly, we established that oxytocin does increase the contractility of uterine horns and that ibuprofen, in large quantities, can inhibit oxytocin induced contractions. Since we were unable to test PGF2a, we can not definitively conclude that PGF2a was the mechanism through which oxytocin produced contractions. However, there is much evidence from other studies to support this claim (Olson, 2003, Parent et al, 2003, Okawa et al, 2001, Strakova et al, 1998, Engstrom, 2002, Nathanielsz, 1978, Mitchell et al, 1998, Thorburn et al, 1979, Chan et al, 1982, Russell et al, 1998). Due to the fact that ibuprofen inhibits the COX-2 enzyme (Chan, 1983, Chan et al, 1982), and in the presence of ibuprofen, contractions were inhibited, we are led to believe that PGF2a is the mechanism of action behind oxytocin induced contractions. We believe our findings support our hypothesis.
APPENDIX A
APPARATUS SET-UP
1. Screw the steel rod into the white, plastic base.
2. Secure the tissue bath clamp to the steel rod, about 15 cm above the base.
3. Secure the glass rod clamp to the steel rod, about 20 cm above the base.
4. Secure the transducer holder directly above the glass rod clamp.
5. Place the tissue bath chamber in the tissue bath clamp.
6. Obtain 8cm of silicone tubing and thread it through the pinch clamp.
7. Attach the 8cm silicon tubing and pinch clamp to the opening at the base of the tissue bath chamber. (*Note – make sure the pinch clamp is closed).
8. Obtain two long tubes of silicone.
9. For the first tube of silicone, attach one end to the thermocycler and the other end to the heated water inlet, located on the lower side of the tissue bath chamber.
10. For the second tube of silicone, attach one end of the tubing to the heated water outlet, located in the middle of the upper side of the tissue bath chamber. Lay the other end of the tubing into the water of the thermocycler.
11. Obtain the oxygen connector and attach one end of the nylon tubing to it.
12. Attach the oxygen connector to the gas inlet, located on the bottom side of the tissue bath chamber, opposite the heated water inlet.
13. Attach the other end of the nylon tubing to the O2-CO2 gas canister.
14. Obtain the transducer and secure it to the apparatus via the transducer holder.
15. Connect the transducer to the iworx data acquisition unit system via channel 3.
16. Calibrate the transducer.
APPENDIX B
ANIMAL CARE
In September of 2006, an Animal Care and Use Committee was established that handled all dealings concerning the ethical treatment of the rats used in this research project. The committee consisted of: Michael Giudici, M.D., Melissa Johnston, R.N., Kirk Kelley, Ph.D., Katherine Matthews, student, Brenda Peters, Ph.D., and Kathy Van Buer, D.V.M. The committee agreed to the following guidelines concerning rat care:
1. Rats are to be kept at room temperature and on 12-hour light and dark periods.
2. Rats are to be caged individually.
3. Injections of any kind should be intramuscular with a 22-25 gauge syringe; all injections should be in the rat’s flank.
4. Food and water should be present ad libitum
5. Euthanization by decapitation using a guillotine is the preferred method.
APPENDIX C
PROTOCOL FOR PHYSIOLOGICAL RINGER’S WITH GLUCOSE
The needed constituents and amounts to make one liter of Ringer’s Solution are as follows: 7.60g NaCl (130mM), 0.45g KCl (6mM), 0.143g MgCl2 (0.7mM), 1.65g NaHCO3 (19.6mM), 0.04g NaH2PO4 (0.29mM), 0.35g Na2HPO4 (1.3mM), 0.44g CaCl2 (3.0mM), and 1.98g D-glucose (11.0mM). To begin, a 3L beaker was rinsed with dilute HCl, followed by distilled water. 1L of distilled water was measure into the 3L beaker. A stir bar was placed into the water and the beaker was placed on a stir plate. All of the above salts were measured out and added to the water in the above order. After addition of each salt, the water was stirred until the salt dissolved. The beaker was then removed from the stir plate and gassed with 95%O2/5%CO2 for a half hour. Sodium hydroxide (NaOH) or hydrochloric acid (HCl) was then added to the solution to obtain a pH of 7.4. The solution was then labeled, parafilmed and stored in the refrigerator until use (Hyland, 2004).
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