Role Of Cns In The Increased Sympathoexcitation In Rats With Fermoral Artery Ligation

Introduction:Peripheral arterial occlusive disease(PAD)is a common cardiovascular disease affected 4?15%adult population.PAD patients exhibit a high coincidence of coronary heart/or cerebrovascular disease(CH/CVD),even though 35?50%of PAD patients without CH/CVD,they all have high morbidity and mortality rates(50-1,500%depending on severity).An exaggerated sympathoexcitation at rest(hypertension)or during physical activity[augmented muscle pressor reflex(MPR)]is found in PAD patients.We propose that the enhanced sympathoexcitation may contribute to this high morbidity and premature death rates in PAD.The mechanisms contributing to augmented CNS(central nervous system)activity in PAD is unknown,but may relate to exaggerated activation of the exercise pressor reflex(EPR).The EPR increases CNS activity and blood preesure(BP)due to afferent neural input arising from contracting muscles to the central CV centers.The brain regions responsible for sympathoexcitation in PAD are unknown but likely involve centers critical to cardiorespiratory regulation.The rostral ventrolateral medulla(RVLM)is essential for tonic SNS activity at rest,and mediates at least a portion of the sympathoexcitatory outflow of the MPR.Nucleus Tractus Solitarius(NTS)is a group of neurons and there is abundant evidence for complex interactions among glutamate,Ang II,GABA and NO.Alterations in PVN function resulting in autonomic dysregulation often involve modulating of these interactions.Furthermore,NTS is a primary center for integrating input signals from baro-,chemo-,and visceral receptors,the excitatory NTS neurons project directly to the direct to PVN,affecting the PVN excitatory output.In this proposed study,we will use a rat model of PAD to test our working hypothesis,and to explore a brand new research field.The outcome from our study will illustrate the regularoty mechanisms contributing to hyper-sympathoexcitation during resting(hypertension)or EPR as found in the PAD patients.The outcome of this study will add to our knowledge for combating PAD and increase the potential to find new therapeutic agents to treat PAD patients.Research Design:1.Animal model establishment:Adult rats weight 275-300g;bilateral femoral artery ligated.Determined after 2weeks.2.Effect of femoral artery occlusion on systemic arterial blood pressure:19 male SD rats,9 received bilateral femoral artery occlusion for 2 weeks,another 10rats services as non-occluded controls.Blood pressure and heart rate of the rat is determined under barbiturate drug(Inactin)anesthesia.300 male SD rats,177 received bilateral femoral artery occlusion for 2 weeks,another123 rats services as non-occluded controls.Blood pressure and heart rate of all the rats is determined before they begin to run on the treadmill.Control(n=26)and Occluded(n=23,2week occlusion)rats.Arterial catheter(PE 50 tube)inserted into left iliac artery for BP monitor.Blood pressure and heart rate of all the rats is determined when they are running on the treadmill at 25m/min.3.Effect of carotid sinus denervation on pressor reflex:Normal(n=6)and Occluded(n=6,2 week occlusion)rats.Carotid sinus denervation study:bilateral carotid arteries were dissected till the internal and external fork,apply phenol locally at fork area to destroy the carotid sinus nerve.Then using 0.1 ml of 10 m M PE bolus iv.Change of blood pressure and heart rate were recorded.4.Effect of proximal sciatic nerve stimulation on systemic blood pressure and RSNA:Normal(n=12)and Occluded(n=11,2 week occlusion)rats,arterial catheter(PE 50 tube)inserted into left iliac artery for BP monitor and RSNA,another catheter inserted into femoral vein for drug delivery,and right sciatic nerve was isolated and cut,the proximal end was connected to a stimulator at different current,with 0.2 ms,60 Hz,and 30 V setting.The renal sympathetic nerve activity(RSNA)is also recorded.Stimulation was stopped when each maximal response on BP and SNA.The stimulation procedures were done before and post carotid sinus denervation(DN).5.Effect of respiration function test in response to hypoxia challenge:Adult male SD rats were divided into control and bilateral femoral artery occluded group(n=4 each group),occlusion for 2 weeks,and placed animal in a whole body plethysmography device to determined respiratory function in response to different oxygen content and high CO₂.Respiration rate,minute ventilation volume,and tidal volume were determined.6.Effect of RVLM in regulating systemic blood pressure in occluded rats:In order to explore the role of RVLM in the augmented sympathetic output,19 rats were used to this study(n=9 for occluded,and n=10 for controls).Rat was under 2%isoflorane anesthesia induction,PE 10 catheters were inserted into right femoral artery for blood pressure and heart rate monitor,and femoral vein for Inactin infusion to maintain under proper anesthesia.Tracheotomy was performed and inserted a tube into the trachea for mechanical ventilation.Rat head was fixed by a sterotactic apparatus,bilateral RVLM was prepared for microinjection the follow agents:Glutamate(Glut)is the major excitatory transmitter(agonist)in RVLM,kynurenic acid(KYN)is an antagonist binding with Glut receptor to block the Glut effect,and inhibiting sympathetic output.We conducted RVLM microinjection to test if the sensitivity of RVLM to Glu has been changed under condition without KYN or with KYN.?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid(AMPA)is an analog of Glut to trig the fast reacting channel of AMPA receptor.7.42 Male adult SD rats were used in this study,14 rats were normal control,the others received femoral artery occlusion(bilateral side=14,single side=14).2 weeks after ligation,rats were anesthetized with Ketamine/Acepromazine.Animals were decapitated,and the brain was dissected out,and frozen under liquid nitrogen chilled isopentane solution,store at-80 degree C freezer.Real-time PCR was used to detect m RNA expression of AT-1?AT-2?SOD-1?CAT?NMDA and n-NOS in the rat's PVN.8.Western Blot was used to detect protein expression of n NOS in the PVN of occluded rats and the normal mentioned in part 7.9.20 Male adult SD rats(weight about 350 g)were used in this experiment.12 rats received femoral artery bilateral occlusion,8 rats were used as normal controls.PVN immunochemical histological(ICH)is examined For Fos and n NOS.Result:1.Hyper sympathoexcitation in rats with femoral occludedAn increased sympathoexcitatory state at rest(hypertension)and during exercise(elevated pressor reflex)found in PAD patients;can be produced in a rat with Bi Fem Occl.Conscious rats receiving 2 weeks Bi Fem Occl exhibit elevated BP from127±2.2(n=26)to 147±2.7(n=23)mm Hg,(P<0.05).In addition,BP during running at25m/min is more than doubled in Bi Fem Occl rats(16mm Hg,n=25)than in normal controls(7±1.7mm Hg,n=25,P<0.05).It implies an increased EPR.2.Furthermore,the BP and Renal sympathetic nerve activity(RSNA)recorded under anesthesia were also higher in rats with Bi Fem Occl.The BP response to excitatory neurotransmitter glutamate RVLM micinjection was similar between the Normal and Bi Fem Occl rats.However,the Bifemoccl rats shown a great BP reduction to glutamate anatagonist kynurenic acid(KYN,40m M).The RT-PCR results showed a remarkmable increase in AR-1R expression(5-fold above normal,P<0.01),moderate increase in SOD-1(4-fold above normal,P<0.01)and NMDA(a fold increase)expression following 3 weeks of Bi Fem Occl.Immunohistochemical(IHC)studies showed that the number of neurons immoreactive with Fos B(a member of inducible transcription factor FOS family)a marker of tonic neuron activation was significantly increased in the PVN following 3 weeks Bi Femoccl(P<0.01,n-6).Yet,the number of n NOS immunoreactive neurons was remakedly reduced in the PVN(P<0.01,n=6)Conclusion:1.Thus,rats with occlusion of bilateral femoral arteries provided a useful animal model to study the exaggerated sympathoexcitation found in PAD patients.2.Increased signal inputs via afferent nerve fibers induce adaptions in the brain cardiovascular(CV)centers,the balance between excitation and inhibiton in the CV centers is reset to a hyper sympathoexcitatory state that increases CV responses,observed during physical activity an likely propagates an augmented sympathoexcitation in PAD patients...