Harrison

Pharmacological Manipulation in the Rat for the Investigation of Hepatic and Cardiorespiratory Physiology.

University of Toronto

Harrison, K. 998854930 March 8th, 2016

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Abstract Here, we have replicated the bromosophalin (BSP) clearance test for hepatic functioning, and cardio-respitory responses to different pharmacological agents. BSPs metabolic half life was estimated at 1.24 min with a plasma clearance of 101.9 mL/kg*min. Pharmacological results indicate the addition of epinephrine increasing heart rate (HR) 28 beats/min (BPM), and mean arterial pressure (MAP) increasing 10mmHg. The addition of Acetylcholine resulted in HR decreasing 36BPM, MAP decreasing 8mmHg. Addition of Atropine resulted in MAP decreasing 10mmHg. Isoproterenol resulted in HR increasing 128/54BPM, 3mmHg increase in PP, and MAP decrease of 9.5mmHg. Finally, nicotine showed a biphasic response, with a HR decrease of 47BPM, PP increase of 1.5mmHg, and decrease in MAP is 10mmHG. Methods of euthanasia showed a gradual decrease in MAP, respiratory rate (RR), and Respiration depth (∆R) with extingunation, a sharp drop off of HR but delayed drop off of BP with KCl, and a cessation of HR and BP shortly after strong respiratory collapse from T61.

Acknowledgements:

Thank you to the coordinators of the PSL374 Module 2 Lab: Dr. J. M. Wojtowicz, and guest speaker Prasad S. Dalvi. Thank you to the Department of Comparative medicine for providing facilities and staff to assist in this investigation. Thank you to the TAs for assisting us, and Lucy Yeung for marking this report. Thank you to the other members of the PSL374 class: Lael Jung, Judi Tran, Hyun Seo, Emma McIlwraith; Louise Nga, Hedy Romero, Cindy Tsui, Risako Kondo ; Yasaman Nahaei, Shaharyar Khuhro, Julia Kim, Vivian Tong; Dylan, Simone, Elaine, Nathan. And special thanks to my partners in this investigation: Carl Swanson, Patrick Gurges, and Bill Yee. I would also like to recognize Luke Dingwell , Henry Ma and Jessica Zung from the 2015 PSL374 class who posted their data for comparison last term.

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Introduction The goal of this module is to to replicate previous findings of hepatic metabolism, and pharmacological substances altering cardiac activity. Here, we use rat as an animal model to familiarize students with surgical manipulations and to experience hepatic and cardiorespiratory physiology. Even though the rat is different from the humans, its physiological processes -for the most part- accurately model humans. Here, investigation of hepatic metabolic clearance utilized the BSP test, which measures the hepatic clearance of BSP over time, and is used to infer: hepatic blood flow (Vh), Plasma cleared (Vp), total blood volume (Vt), BSP half life (t1/2), and the time constant (τ). Additionally, we investigated the cardio-respiratory response to inotropic and chronotropic drugs. An inotropic agent alters the force of muscular contractions, with negative inotropic agents weakening contractions and positive inotropic enhancing contraction, whereas chronotropic agents affect the rate of contraction 8. Pharmacological effects were recorded and analyzed with BioPack software, monitoring blood pressure (BP), Cardiac potential (ECG), and respiration. It is hypothesized that all BSP clearance values should be similar to those presented in Lecture 2 module 2, slides 22-26 14, with our animal showing a linear Ln decrease in BSP concen-tration over time. Additionally, it’s hypothesized that injection of different agents will alter cardiac and respiratory responses indicative of underlying physiology, outlined in (Table 1,2, Fig S15-17).

Materials & Methods Protocols for experimentation are outlined in “PSL-374H 2016 Lab Manual SURGERY I […] II.pdf” provided by the Physiology department of the University of Toronto 14. Surgical set up is shown in Figure 1. Values for drug administration were altered for weights of animals used. Animal weight for lab one was 259g, with 0.143mL of 15mg/uL BSP solution introduced. Animal weight for lab two was 440g, with 0.17mL injections of 2.5ug/uL epinephrine, 2.5ug/uL acetylcholine, 2.5ug/uL atropine, 0.25ug/uL isopantine, and 10ug/uL nicotine. Unfortunately, several deviations from protocol occurred. In Lab 1, canulation of both carotid and jugular artery was disrupted. Due to provided canulae lacking fine tips, the canulae were thus ejected shortly after BSP injection. This resulted in blood loss, and likely a small amount of residual BSP in the system. Canulae were corrected and the experiment repeated. Post-op sample analysis was also disrupted, resulting in a loss of hematocrit values, and time point ‘4 minutes’. Thus, data from other groups have been obtained for comparison and analysis. In Lab 2, experimental animal expired shortly after jugular artery canulation. A secondary 15g heavier animal was provided, however dilutions were based on the first animal’s weight. Thus, amount of drug injected into the animal was less than provided concentration values, but the teaching assistants stated that differences were acceptable. Additionally, ECG and respiratory recordings for control maneuvers were of poorer quality then succeeding recordings, as electrodes were re-adjusted for better signal. Data analysis for lab 2 is absent in the protocol: Calculations for BSP clearance were modelled from PSL374 Module II - Lecture 2 (slides 22-26) 14. BioPack Software was used to analyze traces. Data points were taken between heat beats as indicated by blood pressure trace. Two sets of data points were recorded prior to, post drug treatment, and at points where the general trend of the trace was altered to provide values for BP, PP, BPM, RR, and ∆R.

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Liver Blood Flow Lab: Results and Discussion Results of BSP clearance tests are shown in Table 3, and Figure 2. R2 values for standard curve and Ln(Co) graphs were both greater than 95%. Values of interest were calculated: initial concentration of BSP, Co= 0.054 mg/mL. The fraction of the circulation which flows through the liver/min, K= 0.557. The metabolic half life of BSP, t1/2= 1.24 min. The time constant, inverse of clearance, τ= 1.8. Since the initial volume was 0.1475ml, the volume of distribution was calculated to be 54ml, or 183 mL/kg. Hematocrit was calculated to be 35% and 37%. Thus, total blood volume was calculated at 83.1mL or 281.5 mL/kg. Finally, plasma clearance of BSP was calculated at 30.1 mL/min, or101.9 mL/kg*min, and hepatic blood flow was calculated to be 46.3 mL/ min, or 156.8 mL/ kg*min.

Assumptions: • Amount of BSP used does not create “Saturation” and “Excretion” phases in the BSP curve.

Instead, reticuloendothelial cells did not become oversaturated, and graphical data is linear • BSP is localized to the reticuloendothelial liver cells, and uptake by other tissues is negligible • All BSP present in blood directed to liver will be removed during that pass though the liver • BSP is completely dispersed throughout the blood on immediate injection • White blood cell volume, during hematocrit calculation, is negligible • Reduction in total blood volume VIA sample extractions assumed to not influence calculated

values, such as hepatic blood flow. (Lower hepatic BF would cause lower plasma clearance) • Anaesthesia induced hypothermia is assumed to have minimal effect on results

(though cardiorespiratory systems become progressively more depressed during surgery)

Constants: • k: Amount of blood flowing through the liver at a given unit of time. Amount of dye cleared in

a specified amount of time. • t1/2: Similar to radioactive half-life, pharmacological half-life indicates the time required for

the amount of dye present in the system to be reduced by 50%. • τ: The time constant is an indication as to how well the liver is functioning. It is also the time it

takes to reach 37% of original drug concentration. • Vo: Volume of distribution (assumed to be total volume of plasma in body) or the amount of

dye needed to get the concentrations seen in blood

The results of this test are in agreement with data presented in class (i.e t1/2 in lecture = 1.73min 14; 20% greater than our value) and data obtained from other students (as seen in Table 3, and Figure 2). R2 values indicate a high continuity and consistency of data points. Though it is difficult to state the degree of statistical significance between hematocrit readings, there appears to be significance between hematocrit values. This makes intuitive sense as, though total blood volume is reduced, such blood reduction included both plasma and red blood cells. The calculated plasma volume appears appropriate. Though it is 50% greater than the value shown in lecture14, (20.1mL/min) it can be explained by our lower hematocrit percentage. Thus, greater plasma clearance is likely the result of greater proportion of plasma to hematocrit in our animal.

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Cardiorespiratory Lab: Results and Discussion See Figure 14 for identification of PP, Diastolic pressure, systolic pressure, MAP, RR, and ∆R;

Figure 15 for the cardiac pacemaker and monocyte electric cycle

Saline Injection: Recordings of 1mL showed little effect, with a small indication of depression (~1mmHg) lasting ~25 seconds (data not shown). Saline maneuver was repeated with 4ml, BPM remained 340BPM, with a 1 second 1.4mmHg decrease in MAP; with pulse pressure from PP 1.72 to 1.83, and MAP from 55.6 to 53mmHg (Figure 3). As hypothesized, both the 1mL and 4mL injections should result in a slight increase in BP. This is according to the Frank-Starling Law of the heart: where greater blood volume causes increased pre-load, an increased force of contraction, and then increased BP.

Abdominal Pressure: Application of pressure resulted in HR increasing 49BPM from 326BPM to 375BPM, MAP increasing 1.3mmHg from 56.1 to 57.4mmHg, and PP remaining 1.61mmHg with this effect lasting only as long as compression (Figure 4). This is opposite to my hypothesis, where I predicted a BP decrease from pressure limiting blood flow, reducing venous return, and thus reducing cardiac output (CO). It is then possible that the inverse is happening, where abdominal pressure facilitates venous return instead of inhibiting it.

Chest Pressure: Chest compression resulted in HR increasing 24BPM from 288BPM to 312BPM, MAP decreasing 5.3mmHg from 55.3 to 50mmHg, and PP remaining 2.1mmHg with this effect lasting only as long as compression (Figure 5, Table 1,2). This decrease in BP is likely due to compression reducing pre-load VIA the superior and inferior vena cava, residual after-load from applied thorasic pressure, and an reduction in the space available for atrial and ventricular filling. Thus, end systolic and diastolic pressure would be reduced as there is less blood available to create such pressure. Additionally, it may be that the increase in BPM was the result of compensation for decreased MAP.

Carotid Sinus Reflex: Manipulation of cannulated carotid resulted in HR decreasing 12BPM from 312BPM to 300BPM, MAP decreasing 2.9mmHg from 56.6 to 53.7mmHg, and PP increasing 0.3mmHg with this effect lasting only as long as manipulation (Figure 6, Table 1,2). This response is the result of manipulating the baroreceptors within the carotid artery. Activation of such receptors signals cardiovascular and respiratory control centres leading to decreased parasympathetic activation, norepinephrine (nEPI) release and binding to β1-adrenoceptors, decreased rate of depolarization in the SA node, conduction in the AV node, and contractility of cardiac myocytes leading to decreased HR and BP. Though not observed due to poor trace, it is likely that ∆R increased and RR decreased breathing from this manipulation.

Epinephrine: Introduction of epinephrine (EPI) 2.5 µg/ml into the animal resulted in an interesting trace. We found an initial injection produced a rising phase lasting 4.2 seconds, plateauing for an additional 3.5 seconds. This plateau showed HR increased 28BPM from 312 to 340 BPM, MAP increased 9.9mmHg from 59 to 86mmHg, PP remained at 1.94mmHg during initial injection. Falling phase lasted 84 seconds to a minimal MAP of 47.3mmHG, HR of 312BPM, and PP

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increased 0.5mmHg to 2.4mmHg. Return to baseline required an additional 50 seconds. Interestingly, 200 seconds post exposure, MAP was still elevated at 63mmHg. Respiration and ECG trace showed no alteration of activity (Figure 7, Table 1,2). This response indicates EPI acting as a positive ionotrope, by increasing atrial/ ventricular cardiac muscle contractility and MAP, and a chronotrope, by increasing HR through the Sino atrial (SA) node. The observed transient increase in BP and HR from EPI is likely from it’s direct effect on adrenergic receptors of the heart 5. Activation of such receptors, such as the ß1R on SA pacemaker cells, activates the g-protein coupled receptor (G) Gs pathway 8,15. This leads to protein kinase A (PKA) phosphorylation of phospholamban, which disinhibits L-type Ca2+ channels (See Figure 15 for activity of the SA pacemaker cells, and see Table 1, Table 2 for additional information on this pathway) 8,15. The observed decrease in BP and HR shortly after could be explained by the body counteracting a local sympathetic effect, as presence of epinephrine would normally result from systemic activation of sympathetic chain ganglia. The prolonged increase in MAP and PP over time may be the result of increase total peripheral resistance (TPR) from vasoconstriction mediated by EPI binding to ⍺1 and ⍺2Rs throughout the peripheral vasculature. Unfortunately alterations in ECG and respiration were not observed. Poor ECG readings may be due to improper grounding, placement of electrodes, or interference from the rest wall distorting signal. Respiration trace may have been absent due to the animal’s head placement not being fully in anesthetic apparatus. It is hypothesized that, with a correct reading, EPI would activate ß2Rs in bronchiolar smooth muscle, again activating the Gs pathway, facilitating bronchodilation, and resulting in faster deeper breathing 15.

Acetylcholine: Introduction of acetylcholine (ACh) 2.5 µg/ml showed a short rising phase, with an increase of 3mmHg from 69.8 to 72.8mmHg lasting 6 seconds, and a falling phase lasting 4 seconds to a prolonged depression in which MAP was reduced ~15mmHg from 72.8 to 57.67 mmHg, PP increasing 0.8mmHg from 2.0 to 2.8mmHg, and HR decreasing 37BPM from 394 to 357BPM. This effect lasted ~60 seconds (Figure 8, Table 1,2). The observed effect of ACh is in agreement with my hypothesis. The trace indicates ACh initiating a brief positive chronotropic/ionotroic nicotinic response, and then acting as a prolonged muscarinic negative ionotrope, by reduced contractility and MAP, and a negative chronotrope, by reducing HR through the SA node. ACh’s mechanism of action is through the M2 ACh receptor, where ACh binding activates the Gi pathway 1,5,15. This leads to a decrease in cAMP, and the ßeta-Γ G subunit phosphorylating K+ channels, decreased cAMP production, and increase K+ conductance 1, 15. This also causes decreased activation of the ‘funny current’ channels (If ) 15. This results in the observed reduced HR, and BP. Additionally, ACh is vasodilator through M3Rs. This would reduce TPR and also Lower BP; however, this may be a short term effect, as Nitrous Oxide (NO) is the effector molecule for this vasodilation pathway. Though no respiratory or ECG effects were observed, likely for similar reasons in our EPI trial, it is hypothesized that M3Rs on bronchial smooth muscle would activate Phospho-lipase C (PLC), leading to increased IP3 and DAG singling for bronchoconstriction 8,15. This would result in decreased tidal volume and decreased breathing rate.

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Atropine: Administration of Atropine, 2.5 mg/ml, showed an initial dip in the trace lasting 3.5 seconds, with BP dropping 5mmHg from 65.3 to 60.3mmHg. There was a brief return to baseline for 1 second, and then a secondary reduction in the trace, with BP dropping 10mmHg from 65.3 to 55.5mmHg, PP increasing 0.13mmHg from 2.76 to 2.93mmHg, and HR remaining constant. This effect lasted ~70 seconds. No noticeable effect was observed in respiratory or ECG traces (Figure 9). The observed effect of Atropine is in agreement to my hypothesis. Though Atropine is a mACh antagonist 5, and one night expect antagonistic effects to those of acetylcholine, Atropine however has been observed to produce Bradycardia and effects similar to ACh 15. Here, we see similar a similar response to Atropine as ACh, but without the strong initial nicotinic response. The acquired trace indicates Atropine acting as a negative ionotrope, by reduced contractility and MAP, but not exerting a chronotropic effect, as HR was unaffected. It is possible that Atropine’s effect in producing bradycardia is depend on concentrations not applied in this experiment. For low concentrations, Atropine exerts parasympathomimetic effects15 (refer to ACh for mechanism) . At higher concentrations, atropine acts as a parasympathetic antagonist15. This is possibly due to blockade of mACHRs, and loss of vagal tone on heart. Loss of vagus nerve stimulation on the heart favours sympathetic enervation, increase in HR.

Isoproterenol: Introduction of Isoproterenol 0.25 µg/ml into the circulation resulted in an initial falling phase lasting 30 seconds, and a prolonged MAP depression lasting 50 seconds. During this time, MAP was reduced 10mmHg from 62.9 to 52.2mmHg, HR was increased 127.2BPM from 340 to 468.8 BPM, and PP increased 1.55mmHg from 2.27 to 3.82mmHg. 90 seconds after injection, pressure rose to baseline, with heart rate still evaluated 54BPM at 394BPM, and pulse pressure increased 2.98mmHg to 5.25mmHg. RR remained at ~50BrPM, and ∆R at ~0.01mmHg (Figure 10) As hypothesized, Isoproterenol produced effects similar to EPI 5. Interestingly, PP was much more greatly effected from Isoproterenol injection opposed to epinephrine. This may be because, though Isoproterenol acts on adrenergic receptors similarly to EPI (see epinephrine for pathway), it also activates the Trace amine-associated receptor 1 for additional effects in brain stem regions. Greater increase in PP is possibly due to Isoproterenol having a stronger binding affinity than EPI, thus better activating sympathetic cardio-respatory effects. As in previous results, respiratory data and ECG did not represent effects of this drug.

Nicotine: Finally, addition of Nicotine 50 µg/ml caused a depression from baseline over 10 seconds, with a MAP decrease of 10mmHg from 65.8 to 54.8mmHg, HR decrease of 47BPM from 441.2 to 394BPM, and a 0.5mmHg increase in PP from 4.26 to 4.76mmHg. Then, there was a rising phase lasting 5 seconds in which MAP increased 13mmHg to 66.4mmHg, PP increasing an additional 0.9mmHg to 5.64mmHg, and HR remaining constant. This was then followed by a drawn out depressing lasting >60 seconds, showing MAP again reduced to 54.8mmHg, but with increased PP to 5.8mmHg. (Figure 11, Table 1,2) This trace indicates that Nicotine produces a biphasic, long effect. Nicotine appears to have negative chronotropic effects, by decreasing HR, and varying inotropic effects. The initial decrease in MAP may be from nAChRs mimicking ACh parasympathetic enervation. Over time, Nicotine would increase MAP due to stimulation of autonomic ganglia, as both the Parasympathetic vagal

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ganglia near the heart, and the spinal global sympathetic ganglia are both activated by nAChRs. This would facilitate the release of EPI 8, 16 (chromaffin cells of the adrenal medulla) and nEPI 8, 16

(adrenergic post-ganglionic neurons). Intracellular singling pathways would then be similar to those outlined in EPI. Additionally, evidence indicate bi-phasic responses to Nicotine at the SA node directly, which may also contribute to the observed trace 11. Again, though not observed. it is thought that nicotine would facilitate bronchoconstriction through the production of tachykinins and a cholinergic response.

Euthanasia KCl: Injection of KCL from this year’s group did not illustrate the effect of KCl well, as there was a 12 second delay in the trace 12. Thus a previous year’s trace was used 4. It was found that 1.5 seconds post injection resulted in absence of a heart beat, and MAP reduction from 44 to 9mmHg occurring over ~6 seconds.(Figure 12). Unfortunately, poor trace ECG trace data were unable to illustrate the effect of KCl the absence of heart beats ~1 second after KCl injection makes physiological sense. After heart failure, respiration should stop shortly after 3, but again poor trace recordings failed to illustrate this effect. The observed final pressure, 9mmHg, is likely the mean systemic pressure (MCFP) of the circulation. the reason for cardiac arrest would be depolarization block of cardiac myocytes 3. As seen in Figure 16, K+ conductance is integral for the action of pacemaker cells, and propagation of signal through cardiac myocytes 8. Application of KCl would disrupt the ionic concentrations required for this process.

T61: Application of T61 trace from this year’s group did not illustrate embutramide’s effect well 2. Thus a previous year’s trace was used 4. It was found that RR went from 70 BrPM and ∆R of 3.4mmHg, to a complete loss of RR due to a respiratory spasm. HR ended 1.6 seconds after spasm, and 2.8 seconds after last normal expiration. PP was recorded at 2.3mmHg, HR 394 BPM, and MAP at 50.1mmHg prior to T61 application. Just prior to spasm, MAP was 40mmHg, PP was 5.32mmHg, and HR 326 BPM. MAP flatlined at approximately 20mmHg (Figure 13). T16 is a solution containing the agents Embutramide, which facilitates Respiratory collapse 9, Mebezonium Iodide, to facilitate muscle paralysis and circulatory collapse 9, and Tetracaine Hydrochloride, which is meant to reduces pain at the injection site 5, 9. Similar to reports of this agent 9, respiration ended prior to cardiac arrest. The large spasm seen in the trace is likely the result of Embutramide and Mebezonium Iodide working in tandem. This year’s group trace indicate that the animal was not securely positioned in the respiration apparatus for recordings. The observed final pressure, 20mmHg, is likely the MCFP. The observed increase in MCPF may be explained by Mebezonium Iodide’s action facilitating circulatory collapse.

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Exsanguination: Our group preformed this method 7; however, past year’s data have been used for comparison of results 4. Prior to blood removal, HR was 340 BPM, PP at 1.32mmHg, and MAP 82.36mmHg. Half way through the trial, post 7mL blood removal, HR decreased to 335bpm, and MAP to 49mmHg. After 7mL removal, a working HR trace was unavailable, and a spike in pressure occurred. Thought this process, we removed 18.5mL of blood from the animal (~70% total blood volume), and resulted in a final PP of 0.5mmHg, MAP of 83mmHg, and absent heart beat. Throughout the trace, RR and ∆R decreased. RR went from 35BrPM and ∆R 0.015mmHg to RR of 29.4BrPM and ∆R of 0.006mmHg. A working respiratory trace was lost after 11mL of blood extraction. Last year’s class showed a similar trace, but without a spike in pressure, and absence of working respiratory data (Figure 14). Results of exsanguination are in agreement with hypothesized physiological responses 8. Though we had a large artifact in our data post 7mL extraction, the general trend was in agreement with past class data. The mechanism of death in this animal is similar to the effects of acute hemorrhage on the cardiovascular and respiratory system 8. Reduction in blood volume results in less blood circulating in the system. This would lead to a lower pre-load to the heart, creating a decreased MAP and PP. Loss of blood also equates to loss of oxygen transportation to organs. Thus global tissue hypoxia begins to occur due to lack of nutrients and O2 to tissues. Metabolism of waste products also becomes reduced, due to reduced blood flow to liver and kidneys. Interestingly, additional blood could not be obtained due to negative pressure from circulation, and our animal was still breathing (RR~ 10BrPM) after experimentation. This is likely due to vascular collapse from reduced venous/ arterial pressure. It is likely that our animal survived after such blood loss due to compensatory mechanisms. One such mechanism would be similar to activation of the sympathetic ganglia: in which nEPT and EPI would facilitate vascular constriction.

Conclusion: Here, we have been able to replicate hepatic and cardio-respatory physiological responses expected for such tests. Though some data is inconclusive, as seen in respiration and ECG data, the overall trends in the data are reflective of expected physiological responses.

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References

1. Calloe, K. et al. "Tissue-Specific Effects Of Acetylcholine In The Canine Heart". AJP: Heart and Circulatory Physiology 305.1 (2013): H66-H75. Web.

2. Chan, N., Ngan, L., Yang, J., & Tong, V. (2016). T61 Data. Toronto, ON: University of Toronto.

3. CUKIERMAN, S. "Effects Of Extracellular Potassium On Slow Response Excitability In Rabbit Atrial Trabeculae". Cardiovascular Research 19.12 (1985): 754-761. Web.

4. Dingwell , L., et al. (2015). PSL374 Data. Toronto, ON: University of Toronto.

5. Drugbank.ca. N.p., 2016. Web. 6 Mar. 2016.

6. Gourine, A., and A. V. Gourine. "Neural Mechanisms Of Cardioprotection". Physiology 29.2 (2014): 133-140. Web.

7. Gurges, P., Harrison, K., Swanson, C. & Yee, B.,(2016). PSL374 Data. Toronto, ON: University of Toronto.

8. Hall, John E. Guyton And Hall Textbook Of Medical Physiology. London: Elsevier Health Sciences, 2012. Print.

9. Hellebrekers, L. J. et al. "On The Use Of T61 For Euthanasia Of Domestic And Laboratory Animals; An Ethical Evaluation". Laboratory Animals 24.3 (1990): 200-204. Web.

10. Homma, Ikuo, Hiroshi Onimaru, and Yoshinosuke Fukuchi. New Frontiers In Respiratory Control. New York: Springer, 2010. Print.

11. Ji, S. "Differential Rate Responses To Nicotine In Rat Heart: Evidence For Two Classes Of Nicotinic Receptors". Journal of Pharmacology and Experimental Therapeutics 301.3 (2002): 893-899. Web.

12. Khuhro, S., Kim, J., Nahaei, Y., & Tong, V. (2016). BSP Metabolism Data (KCl). Toronto, ON: University of Toronto.

13. Massey, Cory A. et al. "Mechanisms Of Sudden Unexpected Death In Epilepsy: The Pathway To Prevention". Nature Reviews Neurology 10.5 (2014): 271-282. Web.

14. PSL374H1S Lectures: Module 2. (2016) University of Toronto

15. Rang HP, Dale MM, Ritter JM, Moore PK (2003). "Ch. 10". Pharmacology (5th ed.). Elsevier Churchill Livingstone. p. 139. ISBN 0-443-07145-4.

16. Sala, F., A. Nistri, and M. Criado. "Nicotinic Acetylcholine Receptors Of Adrenal Chromaffin Cells". Acta Physiologica 192.2 (2007): 203-212. Web.

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Tables and Figures:

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Table 2 Summary of chemical receptors relevant to this investigation 1, 8, .

Receptors Responce Effect

⍺1Rs On all vessels → ↑PLC through Gq → ↑IP3 and DAG → ↑Ca2+ release from intracellular stores → ↑Constriction → ↑DBP

Sympathetic vasoconstriction (brain, organs),

⍺2Rs On systemic veins→ ↑cAMP through Gi → ↓MLCP activity; ↓K+ efflux; IP3 channel activation; SR Ca2+ ATPase inactivation→ ↑Ca2+→ constriction of systemic veins→ ↑DBP

vasoconstriction (brain, organs), Inhibition of NT release

ß1Rs On the SA node → ↑cAMP through Gs → ↑cAMP binding to lf channels → ↑Na+ and Ca2+ influx → ↑depolarization rate → ↑HR, ↑BPOn cardiac myocytes → ↑cAMP through Gs → ↑PKA → phosphorylation of voltage-gated Ca2+ channels and phospholamban → ↑voltage-gated Ca2+ channel time open; ↑Ca2+ ATPase on SR → ↑Ca2+ influx; Ca2+ removed faster → ↑contractility; ↓contraction duration

↑SA rate, ↑ AV conduction velocity, ↑ contractility

ß2Rs ↑cAMP through Gs; → ↑activity; ↑K+ efflux; IP3 channel inhibition; sarcoplasmic reticulum Ca2+ ATPase activation → ↑Ca2+ → dilation of veins, skeletal muscle, liver arterioles, and bronchodilation; deeper breathing

Vasodilation (skeletal muscles, veins), Bronchial relaxation

mAChRs M2 on SA node → ↑K+ channels open; ↓cAMP through Gi → ↑K+ efflux; ↓Na+ and ↓Ca+2 influxM3 on vascular endothelial cells → ↑IP3 and DAG → ↑NOM3 on bronchiolar smooth muscle → ↑IP3 and DAG → ↑Ca+2

M2 = ↓HR, ↓contractility, ↓AV conductionM3= ↑Vasodilation, ↑broncho-constriction

nAChRs Located in secondary synapse in autonomic ganglia → ↑Na+ influx → ↑depolarization → ↑release of NTs: parasympathetic ganglia: [↓HR; ↓SBP; ↓DBP from ACh] — Sympathetic [↑HR; ↑SBP; ↑DBP from nEPI and EPI]

Activation ganglia (sympathetic, parasympathetic): Increase (ß4)/ decrease (⍺7) at heart

Table 1: Hypothesized effects of treatments applied during investigation 5,8, .

Sensory Input Pathway HR BP PP Resp. Rate

Resp. Depth

Control Manuvers: • Saline: Increased preload, increased blood volume - - - - -

Abdominal Pressure

• Increased/ decreased TPR from pressure ↓ ↑ - - ↓

Thoracic Pressure • Decreased atrial and ventricular volume available from compression ↑ ↓ - - ↓

Carotid Sinus Reflex

• Manipulation would mimic Hypertension → increase baroreceptor firing rate → decrease in BP

- ↓ - - -

Epinephrine 5 • ß2 receptor → bronchodilation in lungs; ⍺2, ⍺1 peripheral sympathetic effects• ß1 receptors → adrenergic effects on heart’s SA, AV, and myocytes

↑ ↑ ↑ - ↑

Acetylcholine 5 • M3 receptor → IP3 pathway → vasoconstriction (M2,3 Agonist)• Increased sensitivity of chemoreceptors

↓ - - ↑ ↓

Atropine 5 • Block muscarinic receptors (M2,3 Antagonist)• Block ⍺-adrenergic receptors

↑ ↑ - - -

Isoproterenol 5 • Activation of ß1,and ß2, receptor activity similarly to Epinephrine ↑ ↑ ↑ ↑ ↑

Nicotine 5 • Initial cholinergic (m/nAChRs at heart, Parasympathetic ganglia), followed by adrenergic response (Sympathetic ganglia nAChRs, adrenal gland) 11

↑ ↓,↑ -,↑ - -

Standard Curve Preparation Sample Clearance of BSP from Rat (Hematocrit: ~34, 37%)

Standard # (1:100) (mg/mL) AS 580 Sample # Time (Min)

AS 580 Interpolated (mg/mL) Ln(C)

1 0.1 0.15 0.69 1 1 0.480* 0.155 0.104* 0.034 -2.26* -3.38

2 0.08 0.12 0.49 2 2 0.345* 0.056 0.084* 0.014 -2.48* -4.27

3 0.06 0.09 0.39 3 3 0.185* 0.050 0.043* 0.012 -3.15* -4.42

4 0.04 0.06 0.26 4 4 0.100* — 0.023* — -3.77 —

5 0.02 0.03 0.12 5 5 0.060* 0.01 0.015* 0.003 -4.20* -5.81

6 0.01 0.015 0.05 6 6 0.040* 0.01 0.012* 0.002 -4.42* -6.21

Table 3: Results of BSP clearance test spectrophotometry values. Asterix detonates data obtained from another group for comparison. Interpolation was the result of comparison of absorption values from standard against plasma absorption. 7

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Figure 2: Results from BSP clearance test spectrophotometry: standard curve construction for interpolation, and linear representation of BSP clearance/ time. A) Standard curve. Equation and correlation value from standards. B) Rate of BSP clearance over time from two samples. Thanks to 2, 12 for providing comparison data

Calculation of hepatic clearance values are below:LnCo= -2.923 Vi= .1475ml Co= 0.054 mg/mL Vo=54ml => 183 mL/kgK= 0.557 Asume Heme = 35%t1/2= 1.24 Vt= 83.1mL => 281.5 m?/kgτ= 1.8 Vc= 30.1 mL/min => 101.9 mL/kg*min

Vh= 46.3 mL/ min => 156.8 mL/ kg*min

BA

Figure 1: Example of surgical setup for investigation. Note that set-up for BSP-clearance test is similar, but with the pressure transduction system being replaced with an additional syringe to obtain blood samples for spectral analysis A) Surgical for the introduction of various drugs.. B) Surgical set up for extingunation procedure. Similar to A, but with additional carotid canulation.

A B

Removal of Blood during BSP Test

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Figure 3: 4mL injection. 7 Figure 4: Abdominal Pressure. 7

Figure 5: Chest Pressure. 7 Figure 6: Carotid sinus reflex. 7

Figure 7: Results of Epinephrine injection. A) Overview of results. B) Higher magnification of results. Blue marks (top) indicate drug introduction. Grey lines indicate break into later time. 7

A B

Figures 3—15 are results of BioPack data collection. Red indicate Blood pressure readings (mmHg), Blue represents the ECG channel (mA). Green Represents a smoothened respiratory trace (mmHg). Scale bars on figures represent data in respective figure. Artifacts are present in recordings, and are indicated with orange lines in the x axis of respective channel, though likely influence other channels.(Of note, ECG traces are poor especially in control trials, and recordings obtained from other groups as in Fig.13,14)

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BA

Figure 8: Results of Acetylcholine injection. A) Overview of results. B) Higher magnification of results. Blue marks (top) indicate drug introduction. 7

Figure 9: Results of Atropine injection. A) Overview of results. B) Higher magnification of results. Blue marks (top) indicate drug introduction. 7

BA

Figure 10: Results of Isoproterenol injection. A) Overview of results. B) Higher magnification of results. Blue marks (top) indicate drug introduction. 7

BA

Figure 11: Results of Nicotine injection. A) Overview of results. B) Higher magnification of results. Blue marks (top) indicate drug introduction. Grey Lines indicate break into later time. 7

A B

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Figure 14: Results of blood removal injection from this, and previous year's class. A) Results from 2015 PSL347 4. B) Results from 2016 PSL374 7. Marks (top) indicate removal of of 0.5mL blood

_______________________________________________________________________ Note: This diagram appears sufficient in representing the percent blood loss from the rat. Calculations required for question 4.11a are follows:

Blood volume = ~50-70mL/Kg Mass= .455kg V= ~22.7- 31.9 mL Extracted= 18mL Thus, ~60-81% of the animal’s blood was removed

BA

Figure 13: Results of T61 injection from this, and previous year's class. A) Results from 2015 PSL347 4. B) Results from 2016 PSL374 2. Blue marks (top) indicate injection of T61

BA

Figure 12: Results of KCl injection from this, and previous year's class. A) Results from 2015 PSL347 4. B) Results from 2016 PSL374 12. Blue marks (top) indicate injection of KCl

BA

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Figure S. 15: Diagram of different aspects of cardiac and respiration traces.

Figure S. 16: Electrophysiology of the heart. A) SA pacemaker acton potential. B) Cardiac moycyte contraction physiology. 8, 15

BA

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Page � of �17 17

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