Dissertations / Theses on the topic 'Ventrolateral Medulla'

Thesis (M.S.)--University of Missouri-Columbia, 2008.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Includes bibliographical references.

Doctor of Philosophy (PhD)
Little is known about the role that tachykinins, such as substance P and its receptor, the neurokinin-1 receptor, play in the generation of sympathetic nerve activity and the integration within the ventrolateral medulla (VLM) of many vital autonomic reflexes such as the baroreflex, chemoreflex, somato-sympathetic reflex, and the regulation of cerebral blood flow. The studies described in this thesis investigate these autonomic functions and the role of tachykinins through physiological (response to hypercapnoea, chapter 3), anatomical (neurokinin-1 receptor immunohistochemistry, chapter 4) and microinjection (neurokinin-1 receptor activation and blockade, chapters 5 and 6) experiments. In the first series of experiments (chapter 3) the effects of chemoreceptor activation with hyperoxic hypercapnoea (5%, 10% or 15% CO2 in O2) on splanchnic sympathetic nerve activity and sympathetic reflexes such as the baroreflex and somato-sympathetic reflex were examined in anaesthetized rats. Hypercapnoea resulted in sympatho-excitation in all groups and a small increase in arterial blood pressure in the 10 % CO2 group. Phrenic nerve amplitude and phrenic frequency were also increased, with the frequency adapting back to baseline during the CO2 exposure. Hypercapnoea selectively attenuated (5% CO2) or abolished (10% and 15% CO2) the somato-sympathetic reflex while leaving the baroreflex unaffected. This selective inhibition of the somato-sympathetic reflex while leaving the baroreflex unaffected was also seen following neurokinin-1 receptor activation in the rostral ventrolateral medulla (RVLM) (see below). Microinjection of substance P analogues into the RVLM results in a pressor response, however the anatomical basis for this response is unknown. In the second series of experiments (chapter 4), the distribution of the neurokinin-1 receptor in the RVLM was investigated in relation to catecholaminergic (putative sympatho-excitatory “C1”) and bulbospinal neurons. The neurokinin-1 receptor was demonstrated on a small percentage (5.3%) of C1 neurons, and a small percentage (4.7%) of RVLM C1 neurons also receive close appositions from neurokinin-1 receptor immunoreactive terminals. This provides a mechanism for the pressor response seen with RVLM microinjection of substance P analogues. Neurokinin-1 receptor immunoreactivity was also seen a region overlapping the preBötzinger complex (the putative respiratory rhythm generation region), however at this level a large percentage of these neurons are bulbospinal, contradicting previous work suggesting that the neurokinin-1 receptor is an exclusive anatomical marker for the propriobulbar rhythm generating neurons of the preBötzinger complex. The third series of experiments (chapter 5) investigated the effects of neurokinin-1 receptor activation and blockade in the RVLM on splanchnic sympathetic nerve activity, arterial blood pressure, and autonomic reflexes such as the baroreflex, somato-sympathetic reflex, and sympathetic chemoreflex. Activation of RVLM neurokinin-1 receptors resulted in sympatho-excitation, a pressor response, and abolition of phrenic nerve activity, all of which were blocked by RVLM pre-treatment with a neurokinin-1 receptor antagonist. As seen with hypercapnoea, RVLM neurokinin-1 receptor activation significantly attenuated the somato-sympathetic reflex but did not affect the sympathetic baroreflex. Further, blockade of RVLM neurokinin-1 receptors significantly attenuated the sympathetic chemoreflex, suggesting a role for RVLM substance P release in this pathway. The fourth series of experiments (chapter 6) investigated the role of neurokinin-1 receptors in the RVLM, caudal ventrolateral medulla (CVLM), and nucleus tractus solitarius (NTS) on regional cerebral blood flow (rCBF) and tail blood flow (TBF). Activation of RVLM neurokinin-1 receptors increased rCBF associated with a decrease in cerebral vascular resistance (CVR). Activation of CVLM neurokinin-1 receptors decreased rCBF, however no change in CVR was seen. In the NTS, activation of neurokinin-1 receptors resulted in a biphasic response in both arterial blood pressure and rCBF, but no significant change in CVR. These findings suggest that in the RVLM substance P and the neurokinin-1 receptor play a role in the regulation of cerebral blood flow, and that changes in rCBF evoked in the CVLM and NTS are most likely secondary to changes in arterial blood pressure. Substance P and neurokinin-1 receptors in the RVLM, CVLM and NTS do not appear to play a role in the brainstem regulation of tail blood flow. In the final chapter (chapter 7), a model is proposed for the role of tachykinins in the brainstem integration of the sympathetic baroreflex, sympathetic chemoreflex, cerebral vascular tone, and the sympatho-excitation seen following hypercapnoea. A further model for the somato-sympathetic reflex is proposed, providing a mechanism for the selective inhibition of this reflex seen with hypercapnoea (chapter 3) and RVLM neurokinin-1 receptor activation (chapter 5). In summary, the ventral medulla is essential for the generation of basal sympathetic tone and the integration of many vital autonomic reflexes such as the baroreflex, chemoreflex, somato-sympathetic reflex, and the regulation of cerebral blood flow. The tachykinin substance P, and its receptor, the neurokinin-1 receptor, have a role to play in many of these vital autonomic functions. This role is predominantly neuromodulatory.

Thesis (Ph.D.)--University of Mississippi, 2008.
Typescript. Vita. "August 2008." Major professor: Robert C. Speth Includes bibliographical references (leaves 110-128). Also available online via ProQuest to authorized users.

Ventrolateral medullary neurons have important roles in cardiorespiratory coordination. A rostral extension of the ventral respiratory column (RVRC), including the retrotrapezoid nucleus (RTN), has neurons responsive to local perturbations of CO2 / pH. Respiratory-modulated firing patterns of RVRC neurons are attributed to influences of more caudal (CVRC) neurons. These circuits remain poorly understood. This study addressed the hypothesis that both local interactions and influences from the CVRC shape rostral neuron discharge patterns and responses. Spike trains from 294 rostral and 490 caudal neurons were recorded with multi-electrode arrays along with phrenic nerve activity in 14 decerebrate, vagotomized cats. Overall, 214 rostral and 398 caudal neurons were respiratory-modulated; 124 and 95, respectively, were cardiac-modulated. Subsets of these neurons were evaluated for responses to sequential, selective, transient stimulation of central and peripheral chemoreceptors and arterial baroreceptors. In 5 experiments, Mayer wave-related oscillations (MWROs) in neuronal firing rates were evoked, enhanced, or reduced following central chemoreceptor stimulation. Overall, 174 of the rostral neurons (59.5%) had short- time scale correlations with other RVRC neurons. Of these, 49 triggered cross-correlograms with RVRC targets yielding 330 offset features indicative of paucisynaptic actions from a total of 2,884 rostral pairs evaluated. Forty-nine of the CVRC neurons (10.0%) were triggers in 142 CVRC-RVRC correlograms - from a total of 8,490 - with offset features indicative of actions on RVRC neurons. Correlation linkage maps support the hypothesis that local circuit mechanisms contribute to the respiratory and cardiac modulation of RVRC neurons and their responses to chemoreceptor and baroreceptor challenges.

Doctor of Philosophy(PhD)
This thesis addresses the neurochemistry and function of specific nuclei in the autonomic nervous system that are crucial mediators of cardiorespiratory regulation. The primary aim is to build on previous knowledge about muscarinic cholinergic mechanisms within cardiorespiratory nuclei located in the ventrolateral medulla oblongata. The general focus is characterisation of gene expression patterns of specific muscarinic receptor subtypes in central nuclei involved in blood pressure control and respiratory control in normal rats. The findings were subsequently extended by characterisation of muscarinic receptor gene expression patterns in 1) a rat model of abnormal blood pressure control (hypertension) (Chapter 3) 2) a rat model of cholinergic sensitivity (Chapter 5) 3) the rat ventral respiratory group (Chapter 6) The results of a series of related investigations that ensued from the initial aims more finely characterise the neurocircuitry of the ventrolateral medulla, from a specifically cholinoceptive approach. All five muscarinic receptor subtypes are globally expressed in the ventrolateral medulla but only the M2R mRNA was significantly elevated in the VLM of hypertensive animals compared to their normotensive controls and in the VLM of animals displaying cholinergic hypersensitivity compared to their resistant controls. Surprisingly, M2R mRNA is absent in catecholaminergic cell groups but abundant in certain respiratory nuclei. Two smaller projects involving gene expression of other neurotransmitter / neuromodulators expressed in cardiorespiratory nuclei were also completed during my candidature. Firstly, the neurochemical characterisation of enkephalinergic neurons in the RVLM, and their relationship with bulbospinal, catecholaminergic neurons in hypertensive compared to normotensive animals was carried out (Chapter 4). A substantial proportion of sympathoexcitatory neurons located in the RVLM were enkephalinergic in nature. However, there was no significant difference in preproenkephalin expression in the RVLM in hypertensive compared to normotensive animals. Secondly, the identification and distribution of components of the renin-angiotensin aldosterone system (RAAS) within the brainstem, and differences in gene expression levels between hypertensive and normotensive animals was also investigated. The RAAS data was not included in this thesis, since the topic digresses substantially from other chapters and since it is published (Kumar et al., 2006). The mRNA expression aldosterone synthase, mineralocorticoid receptor (MR1), 12-lipoxygenase (12-LO), serum- and glucocorticoid- inducible kinase and K-ras) were found to be present at all rostrocaudal levels of the ventrolateral medulla. Expression of MR1 mRNA was lower in the RVLM of SHR compared with WKY rats and 12-LO mRNA levels were lower in the CVLM in SHR compared with WKY rats. Otherwise, there was no difference in gene expression level, or the method of detection was not sensitive enough to detect differences in low copy transcripts between hypertensive and normotensive animals.

Obesity afflicts more than 30% of the U.S. population and is a major risk factor for the development of hypertension, type II diabetes, and cardiovascular disease. Studies in humans and animals indicate that obesity is associated with increased sympathetic outflow to the vasculature and kidneys. One mechanism postulated to underlie the increase in sympathetic nerve activity (SNA) in obesity is hyperinsulinemia. Little is known regarding the central circuitry underlying elevated SNA and arterial blood pressure (ABP) during hyperinsulinemia and obesity or if sympathoexcitatory circuits are still responsive to insulin in obesity. Hyperinsulinemic-euglycemic clamps elevate SNA to the hind limb vasculature in lean rodents but obesity is associated with resistance to the peripheral and anorexic effects of insulin. Therefore, the first aim was to determine whether diet-induced obesity causes development of insulin resistance in the central circuits mediating SNA. The sympathoexcitatory response to insulin was still intact in diet-induced obese rats indicating a role for insulin in the elevation in SNA and ABP in obesity. The second aim of this project was to identify the specific receptors in the rostral ventrolateral medulla (RVLM) that mediate the elevated SNA during hyperinsulinemia. The RVLM provides basal sympathetic tone and maintains baseline ABP. Glutamate is the major excitatory neurotransmitter and glutamate receptors of the RVLM are known to mediate multiple forms of hypertension. Blockade of RVLM NMDA-specific glutamatergic receptors reverses the increased lumbar SNA associated with hyperinsulinemia. In contrast, blockade of angiotensin II type 1 or melanocortin receptors in the RVLM had no effect on the sympathoexcitatory response to insulin. The goal of the third aim was to identify the cellular mechanisms within RVLM that mediate the elevated SNA and ABP in diet-induced obesity. Blockade of RVLM glutamate receptors reversed the elevated ABP and lumbar SNA associated with diet-induced obesity while it had no effect on rats on a low fat diet or those resistant to weight gain on the high fat diet. Similar to the findings during hyperinsulinemia, blockade of RVLM angiotensin II type 1 or melanocortin receptors had no effect on lumbar SNA or ABP during diet-induced obesity.

The hypothalamic paraventricular nucleus (PVN) is known to be a major integrative region within the forebrain. It is composed of functionally different subgroups of neurons, including the parvocellular neurons that project to important autonomic targets in the brainstem e.g. the rostral ventrolateral medulla (RVLM) and the intermediolateral cell column (IML) of the spinal cord, where the sympathetic preganglionic motor-neurons are located. These regions are critical in cardiovascular regulation; hence, these projections are likely to mediate the effects of the PVN on sympathetic nerve activity and hence may contribute to the cardiovascular changes induced by physiological stimuli such as elevations in body temperature. The neurotransmitter such as nitric oxide (NO) is important in cardiovascular regulation and it is now emerging as a major focus of investigation in thermoregulation. One of the most striking accumulations of NO containing-neurons is in the PVN where it appears to be playing an important role in cardiovascular regulation and body fluid homeostasis. The results of the work show; 1. That spinally-projecting and nitrergic neurons in the PVN may contribute to the central pathways activated by exposure to a hot environment. 2. Suggests that nitrergic neurons and spinally- projecting neurons in the brainstem may make a small contribution to the central pathways mediating the reflex responses initiated by hyperthermia. 3. The present study also illustrates that these PVN neurons projecting to the RVLM may make a smaller contribution than the spinal-projecting neurons in the PVN to the cardiovascular responses initiated by heat. 4. The results of my studies showed that the microinjection of muscimol to inhibit the neuronal activity in the PVN abolished the reflex decrease in renal blood flow following an elevation of core body temperature. In addition, this effect was specific to the PVN, since microinjections of muscimol into areas outside the PVN were not effective. These findings demonstrate that the PVN is critical for this reflex cardiovascular response initiated by hyperthermia. In conclusion, PVN is critical for the reflex decrease in renal blood flow during elevations in core body temperature. We hypothesise that projections from the PVN to the spinal cord and the RVLM contribute to the reflex cardiovascular responses. Additionally, nitrergic neurons in the PVN may contribute but the physiological role of those neurons in the reflex responses elicited by hyperthermia needs to be investigated.

Nesse estudo investigamos os efeitos da hipóxia tecidual aguda (HA) sobre as propriedades eletrofisiológicas intrínsecas dos neurônios pré-simpáticos bulboespinhais da área rostro-ventrolateral do bulbo (RVLM) de ratos jovens adultos submetidos previamente à hipóxia crônica intermitente (HCI) e os seus respectivos controle. Para marcarmos os neurônios pré-simpáticos bulboespinhais da RVLM, ratos Wistar jovens (P19-P21) anestesiados com ketamina e xilazina, receberam microinjeções bilaterais de rodamina, um traçador fluorescente retrógrado, na coluna intermediolateral da medula espinhal (T3-T6) e 2 dias após a recuperação da cirurgia, os animais foram submetidos ao protocolo de HCI, enquanto que ratos controle foram mantidos em condições de normóxia, durante 10 dias. No décimo primeiro dia, os ratos foram novamente anestesiados para a remoção do cérebro e as fatias do tronco cerebral contendo neurônios pré-simpáticos com marcação positivas foram registrados. Utilizamos a técnica de whole cell patch-clamp para estudo das propriedades eletrofisiológicas desses neurônios. As propriedades eletrofisiológicas intrínsecas foram analisadas antes e após a HA, a qual foi produzida pela perfusão das fatias do tronco cerebral com uma solução hipóxica (95% N2 + 5% CO2) durante 2 minutos na presença de bloqueadores sinápticos excitatórios e inibitórios. Todos os neurônios pré-simpáticos apresentaram característica intrínseca de autodespolarização e a frequência de disparos basal de potenciais de ação (PAs) desses neurônios de ratos do grupo controle e HCI foram similares [Controle= 5,03 ± 0,4 Hz (n=39) vs HCI= 6,31 ± 0,7 Hz (n=31); p > 0,05]. No grupo controle, a HA não alterou a frequência média de disparos de PAs (BS = 5,03 ± 0,4 Hz vs HA = 5,24 ± 0,3 Hz (n=39); p > 0,05], porém revelou diferentes perfis de disparo de PAs após 2 min de exposição à HA: i) 11 neurônios com aumento na frequência de disparos (BS = 5,1 ± 0,7 Hz vs HA = 7 ± 0,7 Hz; p < 0,05]; ii) 21 neurônios sem alteração na frequência de disparos (BS = 4,8 ± 0,5 Hz vs HA = 5,36 ± 0,6 Hz; p > 0,05] e iii) 7 neurônios com diminuição na frequência de disparos (BS = 7,3 ± 1,1 Hz vs HA = 3,6 ± 0,7 Hz; p < 0,05). No grupo HCI, a HA produziu aumento na frequência média de disparos (BS= 6,31 ± 0,7 Hz vs HA= 7,25 ± 0,8 Hz; n=31 - p < 0,05) e na análise do perfil de disparo de PAs, a HA revelou 2 subpopulações: i) 9 neurônios com aumento na frequência de disparos (BS = 4,7 ± 0,8 Hz vs HA = 8,2 ± 1,4 Hz; p < 0,05) e ii) 22 neurônios sem alteração na frequência de disparos (BS = 7,0 ± 1,0 Hz vs HA = 6,8 ± 1,0 Hz; p > 0,05). Esse estudo nos permitiu revelar diferentes subpopulações de neurônios pré-simpáticos que responderam de forma distintas à HA. Os resultados também sugerem que a HCI teria um efeito pré- condicionante na excitabilidade intrínseca dos neurônios pré-simpáticos em resposta à HA
In this study we evaluated the effects of acute hypoxia (AH) on the intrinsic electrophysiological properties of presympathetic neurons from rostro ventrolateral medulla (RVLM) of juvenile rats exposed to chronic intermittent hypoxia (CIH) or normoxic condition (control group). To label the RVLM bulbospinal presympathetic neurons, young Wistar rats (P 19 - 21) anesthetized with ketamine and xylazine, received bilateral microinjections of a fluorescent retrograde tracer (rhodamine retrobeads) were performed into the intermediolateral column of spinal cord (T3-T6) and two days after recovery of the surgery, the animals were submitted to CIH or normoxic protocol, during 10 days. On the 11th day, under anesthesia, brainstem slices were obtained and only the labeled RVLM presympathetic neurons were recorded, using whole-cell patch-clamp approach to study the electrophysiological properties of these neurons. The intrinsic electrophysiological properties were analyzed before and after AH, which was produced by slice perfusion with hypoxic solution (95% N2 and 5% CO2) during 2 min in the presence of excitatory and inhibitory synaptic antagonists. All recorded RVLM presympathetic neurons presented intrinsic pacemaker activity and the baseline firing frequency of these neurons from control and CIH group were similar [Control= 5,03 ± 0,4 Hz (n=39) vs HCI= 6,31 ± 0,7 Hz (n=31); p > 0,05]. In the control group, AH do not change the firing rate (BS = 5,03 ± 0,4 Hz vs HA = 5,24 ± 0,3 Hz (n=39); p > 0,05), but revealed different pattern of firing frequency after 2 min of AH: i) 11 neurons increased the firing frequency (BS = 4,9 ± 0,9 Hz vs HA = 6,9 ± 1,0 Hz; p < 0,05) ; ii) 21 neurons do not change the firing frequency (BS = 4,8 ± 0,5 Hz vs HA = 5,36 ± 0,6 Hz; p > 0,05) and iii) 7 neurons decreased the firing frequency (BS = 7,3 ± 1,1 Hz vs HA = 3,6 ± 0,7 Hz; p < 0,05). In the CIH group, the AH increased the firing rate comparing with basal condition (SB= 6,31 ± 0,7 Hz vs AH= 7,25 ± 0,8 Hz; n=31 - p < 0,05) and analyzing the pattern of action potential, AH revealed 2 subpopulations in this group: i) 9 neurons increased the firing frequency (SB = 4,7 ± 0,8 Hz vs AH = 8,2 ± 1,4 Hz; p < 0,05) and ii) 22 neurons do not change the firing frequency (SB = 7,0 ± 1,0 Hz vs AH = 6,8 ± 1,0 Hz; p > 0,05).. The data shows that AH revealed different subpopulations of presympathetic neurons and suggest that CIH plays a preconditioning in the intrinsic excitability of presympathetic neurons in response to acute hypoxia

Made available in DSpace on 2015-07-22T20:50:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-01-28. Added 1 bitstream(s) on 2015-08-11T03:25:41Z : No. of bitstreams: 1 Publico-086a.pdf: 1000885 bytes, checksum: a8256b60ac3216d579a9701c84515583 (MD5). Added 1 bitstream(s) on 2015-08-11T03:25:41Z : No. of bitstreams: 2 Publico-086a.pdf: 1000885 bytes, checksum: a8256b60ac3216d579a9701c84515583 (MD5) Publico-086b.pdf: 507734 bytes, checksum: 35a71879a83bad776c76020f76fb96ee (MD5). Added 1 bitstream(s) on 2015-08-11T03:25:41Z : No. of bitstreams: 3 Publico-086a.pdf: 1000885 bytes, checksum: a8256b60ac3216d579a9701c84515583 (MD5) Publico-086b.pdf: 507734 bytes, checksum: 35a71879a83bad776c76020f76fb96ee (MD5) Publico-086c.pdf: 1776030 bytes, checksum: d420deb32e6a60ac8db7b56dccce3684 (MD5)
Respostas cardiovasculares podem ser integradas em diferentes níveis do sistema nervoso central (SNC). No bulbo existem várias áreas importantes para o controle cardiovascular. Sabe-se que, na superfície ventrolateral do bulbo, existem pelo menos três regiões relevantes ao controle cardiovascular: região rostroventrolateral (RVL), região caudoventrolateral (CVL) e a área pressora caudal (APC). A região RVL tem importante função na manutenção do tônus vasomotor e a região CVL interfere na regulação da pressão arterial (PA) por modular a atividade da região RVL. Além disso, essas duas regiões participam na interação de reflexos cardiovasculares, que incluem a participação de outra estrutura bulbar (Núcleos do Trato Solitário - NTS) como primeira sinapse de fibras aferentes reflexas. A APC foi recentemente descoberta e interfere na regulação da PA por modular a atividade dos neurônios das regiões RVL e CVL. Desse modo, o presente estudo procurou investigar a participação do NTS (região comissural e intermediária) nas respostas produzidas pela estimulação ou inibição da APC. Foram utilizados ratos Holtzman (280-350 g; n = 6-8) anestesiados com uretana (1,2 g/kg, i.v.). Um cateter foi inserido na artéria e veia femoral desses animais para registro de pressão arterial, freqüência cardíaca e injeção de drogas. Em alguns animais, foi registrada a atividade elétrica do nervo esplâncnico e do nervo frênico. Os animais foram adaptados a um aparelho estereotáxico e submetidos a uma craniotomia parcial para injeções de drogas. Num primeiro grupo experimental, os animais foram submetidos a lesão fictícia ou eletrolítica (1 mA x 10s) do NTS comissural (NTScom). A injeção unilateral do aminoácido excitatório L-glutamato (1 mM - 100 nl) na APC produziu resposta pressora (D = +28 ± 3 mmHg, vs. controle: D = +2 ± 2 mmHg, p<0,05) e taquicárdica (D = +20 ± 1 bpm, vs. controle: D = +3 ± 3 bpm, p<0,05). As respostas pressora e taquicárdica promovidas pela injeção unilateral de L-glutamato na APC foram abolidas 10 (D = +5 ± 7 mmHg e D = +4 ± 3 bpm, respectivamente), 30 (D = +7 ± 3 mmHg e D = +6 ± 3 bpm, respectivamente) e 60 minutos (D = +11 ± 3 mmHg e D = +5 ± 1 bpm, respectivamente) após a lesão eletrolítica do NTScom. Da mesma maneira, no grupo de animais que recebeu a injeção do agonista GABA-A muscimol no NTScom, as respostas pressora e taquicárdica promovidas pela injeção unilateral de L-glutamato na APC foram abolidas 10 (D = +5 ± 5 mmHg e D = +5 ± 3 bpm, respectivamente), 30 (D = +8 ± 5 mmHg e D = +7 ± 2 bpm, respectivamente) e 60 minutos (D = +11 ± 6 mmHg e D = +5 ± 2 bpm, respectivamente) após a injeção de muscimol. Por outro lado, as respostas pressora e taquicárdica promovidas pela injeção unilateral de L-glutamato na APC não foram modificadas 10 (D = +11 ± 2 mmHg e D = +30 ± 12 bpm, respectivamente), 30 (D = +27 ± 4 mmHg e D = +32 ± 15 bpm, respectivamente) e 60 minutos (D = +24 ± 7 mmHg e D = +24 ± 8 bpm, respectivamente) após as injeções bilaterais de muscimol no NTS intermediário. Dessa forma, as repostas de estimulação da APC parecem depender da integridade dos neurônios localizados no NTScom. Sendo assim, será que a integridade da APC é importante para as respostas de estimulação do NTScom? Apenas as injeções bilaterais do agonista GABAérgico muscimol (2 mM - 100 nl) na APC promoveu queda de PAM (D = -18 ± 6 mmHg, p<0,05) e FC (D = - 21 ± 8 bpm, p<0,05). As respostas depressora e bradicárdica promovidas pela injeção unilateral de L-glutamato no NTScom foram abolidas 10 (D = -5 ± 3 mmHg e D = +8 ± 9 bpm, respectivamente), 30 (D = -5 ± 2 mmHg e D = +7 ± 4 bpm, respectivamente) e 60 minutos (D = -6 ± 4 mmHg e D = +10 ± 6 bpm, respectivamente) após as injeções bilaterais de muscimol na APC. Da mesma maneira do ocorrido com as respostas de estimulação da APC, as respostas depressora e bradicárdica promovidas pela injeção unilateral de L-glutamato no NTS intermediário não foram modificadas 10 (D = -41 ± 9 mmHg e D = -67 ± 26 bpm, respectivamente), 30 (D = -31 ± 12 mmHg e D = -23 ± 4 bpm, respectivamente) e 60 minutos (D = -32 ± 8 mmHg e D = -21 ± 3 bpm, respectivamente) após as injeções bilaterais de muscimol na APC. Nossa próxima questão era saber se a inibição da APC promoveria alterações nos reflexos cardiovasculares. A curva do barorreflexo foi construída com injeções endovenosas de uma dose depressora de nitroprussiato de sódio (30 mg/kg) e com a inflação de um balão na aorta torácica. A relação de atividade simpática e PAM (curva do barorreflexo) não revelou nenhum efeito significativo alterado após as injeções bilaterais de muscimol na APC. Após a inibição da APC o barorreflexo continuou operando nos mesmos valores de PAM50 quando comparado ao grupo controle. Os valores de platô superior e inferior, bem como o ganho também não tiveram diferença quando comparados com o grupo controle. A ativação do quimiorreflexo foi realizada com injeções endovenosas de cianeto de sódio (NaCN – 50 mg/kg). A ativação do quimiorreflexo com NaCN produziu resposta pressora (D = +16 ± 4 mmHg, vs. salina D = +2 ± 2 mmHg, p<0,05), aumento da atividade simpática do nervo esplâncnico (D = +236 ± 11% em relação ao controle, p<0,01) e aumento da atividade do nervo frênico. Muscimol injetado bilateralmente na APC promoveu uma redução da resposta pressora (D = +9 ± 2 mmHg, vs. salina D = +18 ± 4 mmHg, p<0,05), da resposta simpatoexcitatória (D = +88 ± 18%, vs. salina D = +217 ± 33 %, p<0,01) e do aumento da atividade do nervo frênico produzido pela ativação dos quimiorreceptores periféricos com NaCN. As aferências dos quimiorreceptores periféricos terminam predominantemente no NTScom. Para corroborar a interação existente entre os neurônios do NTScom e a APC, o próximo experimento foi realizado para testar se os neurônios localizados no NTS ou na APC, ativados por hipóxia, são predominantemente glutamatérgicos ou gabaérgicos. Os animais foram expostos a situações de hipóxia (8% O2, n = 5) ou normóxia (ar ambiente, n = 4). A imunorretividade para a proteína Fos foi utilizada para identificação dos neurônios ativados por hipóxia. Os neurônios glutamatérgicos ou gabaérgicos foram definidos devido à expressão, via hibridzação in situ, de VGLUT2 mRNA ou GAD67 mRNA, respectivamente. A maioria dos neurônios que expressam Fos após hipóxia são glutamatérgicos na região do NTScom (64 ± 13%) e NTS intermediário (41 ± 9%). Na região da APC, dos neurônios que expressam Fos após hipóxia, somente uma pequena parcela (26 ± 9%) são glutamatérgicos. Apenas uma pequena quantidade de neurônios que expressam Fos também expressam GAD67 mRNA na região do NTScom (7 ± 4%), na região do NTS intermediário (16 ± 9%) e na APC (5 ± 3%). A imunorreatividade para a proteína Fos foi praticamente inexistente nos animais que foram expostos ao ar ambiente. Diante disso, os nossos resultados são os primeiros a mostrar a existência da interação entre a região comissural do NTS e a APC no que diz respeito ao controle cardiovascular. Sabe-se que a região do NTScom contém neurônios de segunda ordem que recebem as aferências dos quimiorreceptores periféricos e que as respostas de estimulação do NTScom parecem depender da integridade da APC. Logo, os resultados do presente estudo também mostraram que a inibição da APC foi capaz de reduzir as respostas simpatoexcitatórias e respiratórias após a estimulação dos quimiorreceptores periféricos. Por outro lado, a inibição da APC não foi capaz de reduzir as respostas de ativação do barorreflexo.
Cardiovascular responses are integrated at different levels of the central nervous system (CNS). In the brainstem, there are different areas related to the cardiovascular control. It is well known that in the ventrolateral medulla there are, at least, three important regions involved with cardiovascular control: the rostral ventrolateral medulla (RVLM), the caudal ventrolateral medulla (CVLM) and the caudal pressor area (CPA). The RVLM is the main source of excitatory input to sympathetic preganglionic neurons playing a crucial role for tonic and reflex control of the cardiovascular system. The caudal ventrolateral medulla (CVLM) contains the GABAergic sympathoinhibitory neurons that project to RVLM. Besides these two regions, the NTS is the site of the first synapse of the viscerosensory afferent fibers in the brainstem including those related to cardiovascular and respiratory afferents. Besides the circuitry described above, more recent studies have also shown a new site, the CPA, located in the caudal end of the ventrolateral medulla, which provides excitatory signals that affect sympathetic activity to the cardiovascular system in rats. Therefore, the aim of the present study was to investigate the involvement of the NTS (commissural and/or intermediate regions) on the stimulation or inhibition of the CPA. Male Holtzman rats (280-350 g; n = 6-8) anesthetized with urethane (1.2 g/kg, i.v.) were used. Unilateral microinjection of L-glutamate (1 mM - 100 nL) into the CPA resulted in an increase in mean arterial pressure (MAP, D = +28 ± 3 mmHg, vs. controle: D = +2 ± 2 mmHg, p<0.05) and heart rate (HR, D = +20 ± 1 bpm, vs. controle: D = +3 ± 3 bpm, p<0.05). Electrolytic lesions of the commissural NTS (commNTS) (1 mA x 10s) abolished the pressor and tachycardic responses at 10 (D = +5 ± 7 mmHg and D = +4 ± 3 bpm), 30 (D = +7 ± 3 mmHg and D = +6 ± 3 bpm) and 60 minutes (D = +11 ± 3 mmHg and D = +5 ± 1 bpm) produced by injection of L-glutamate into the CPA. In the same way, muscimol (GABA-A receptor agonist) (2 mM - 60 nL) injected into the commNTS abolished the pressor and tachycardic responses at 10 (D = +5 ± 5 mmHg and D = +5 ± 3 bpm), 30 (D = +8 ± 5 mmHg and D = +7 ± 2 bpm) and 60 minutes (D = +11 ± 6 mmHg and D = +5 ± 2 bpm) produced by injection of L-glutamate into the CPA. On the other hand, the injection of Lglutamate unilaterally into the CPA 10, 30 and 60 minutes after the injection of muscimol into the intermediate NTS produced no change on MAP increase (D = +11± 2, +27 ± 4 and +24 ± 7 mmHg, respectively) and HR (D = +30 ± 12, +32 ± 15 and +24 ± 8 bpm, respectively). It seems that the activation of the CPA depends on the integrity of the commNTS neurons. Does the CPA integrity depend on NTS activation? Bilateral injection of muscimol (GABA-A agonist, 2 mM – 60 nl) into the CPA produced a decrease in MAP (D = -18 ± 2 mmHg, p<0.05) and HR (D = -21 ± 6 bpm, p<0.05). The depressor and bradycardia responses produced by L-glutamate into the commNTS was abolished at 10 (D = -5 ± 3 mmHg and D = +8 ± 9 bpm), 30 (D = -5 ± 2 mmHg and D = +7 ± 4 bpm) and 60 minutes (D = -6 ± 4 mmHg and D = +10 ± 6 bpm) after muscimol injection into the CPA. On the other hand, the depressor and bradicardic responses produced by L-glutamate into the intermediate NTS was not changed at 10 (D = -41 ± 9 mmHg and D = -67 ± 26 bpm), 30 (D = -31 ± 12 mmHg and D = -23 ± 4 bpm) and 60 minutes (D = -32 ± 8 mmHg and D = -21 ± 3 bpm) after muscimol injection into the CPA. Our next question was to know if the inhibition of the CPA will change the cardiovascular reflexes. Baroreflex curves were generated by lowering MAP with sodium nitroprusside (30 mg/kg) and increasing MAP by constricting an abdominal aortic snare. The relationship between MAP and sympathetic nerve discharge (SND) (baroreflex curves) revealed no change after muscimol injection into the CPA. After inhibition of the CPA, the baroreflex operated around a comparable MAP (MAP50) in all groups of rats. The lower and upper plateau, as well the baroreflex range was not different from the control group. The chemoreflex was actvated by i.v. injections of sodium cyanide (NaCN – 50 mg/kg). The activation of chemoreflex with NaCN produced a rise in MAP (D = +16 ± 4 mmHg, vs. saline D = +2 ± 2 mmHg, p<0.05), an increase in SND (D = +236 ± 11% of resting, p<0.01) and an increase in phrenic nerve discharge (PND). Muscimol injection into the CPA reduced the pressor (D = +9 ± 2 mmHg, vs. saline D = +18 ± 4 mmHg, p<0.05), sympathoexcitatory (D = +88 ± 18%, vs. saline D = +217 ± 33 %, p<0.01) and the increase in PND produced by chemoreflex activation with NaCN. Carotid chemoreceptor afferents terminate predominantly in commNTS. The next experiment was designed to test and to confirm the interaction between the commNTS and the CPA neurons by anatomical approaches. We tested whether glutamatergic or GABAergic. The rats were exposed to hypoxia (8% O2, n = 5) or to normal air (n = 4). Fos-immunoreactivity was used to identify commNTS neurons that were activated by hypoxia. NTS or CPA neurons were classified as glutamatergic or GABAergic based on whether they contained VGLUT2 mRNA or GAD67 mRNA. The vast majority of the commNTS (64 ± 13%), intermediate NTS (41 ± 9%) and CPA (26 ± 9%) neurons that were immunoreactive for Fos contained VGLUT2 mRNA, whereas only a small proportion of the same class of neurons contained GAD-67 mRNA (commNTS: 7 ± 4%; intermediate NTS: 16 ± 9% and CPA: 5 ± 3%). Therefore, our results are the first to show the existence of an interaction between the commNTS and CPA in terms of cardiovascular control. However we know that commNTS neurons receive inputs from peripheral chemoreceptors and the stimulation of commNTS depends on CPA integrity. These data suggest that CPA contributes to resting MAP, sSND and to the sympathoexcitatory effect during stimulation of peripheral chemoreceptors.
TEDE
BV UNIFESP: Teses e dissertações

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-08-09T11:39:12Z No. of bitstreams: 2 Dissertaçao - Lara Marques Naves - 2018.pdf: 4245553 bytes, checksum: 32754e93d07b1f96bc7f0b9a2bc618ff (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)
Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-08-09T12:20:22Z (GMT) No. of bitstreams: 2 Dissertaçao - Lara Marques Naves - 2018.pdf: 4245553 bytes, checksum: 32754e93d07b1f96bc7f0b9a2bc618ff (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)
Made available in DSpace on 2018-08-09T12:20:22Z (GMT). No. of bitstreams: 2 Dissertaçao - Lara Marques Naves - 2018.pdf: 4245553 bytes, checksum: 32754e93d07b1f96bc7f0b9a2bc618ff (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-03-02
Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
Hemodynamic and cardiovascular benefits from the hypertonic saline solution (HS) use in the hypotensive hemorrhage (HH) treatment have been reported for several years. Recent investigations have shown the participation of central nervous system (CNS) regions, such as A1 neuronal clusters (located in the caudal ventrolateral medulla; CVLM), A2 neuronal clusters (located in the nucleus of the solitary tract; NTS) and the Median Preoptic Nucleus (MnPO) on hemodynamic responses induced by sodium chloride overload in normovolemic animals. However, the role of the above structures in cardiovascular recovery and autonomic changes induced by HS solution administration in animals submitted to HH has not yet been evaluated. Thus, the present study evaluated the A1, A2 neuronal clusters and MnPO nucleus involvement in the cardiovascular and autonomic responses promoted by HS solution infusion in hypovolemic animals. For this, wistar rats (280-320 g) were separated into four protocols: I. A2 neuronal cluster lesion (A2 Sham: n = 6; A2 Experimental: n = 6); II. A1 neuronal cluster lesion (A1 Sham: n = 6; A1 Experimental: n = 6); III. A1 and A2 neural clusters concomitant lesions (A1 + A2 Sham: n = 6; A1 + A2 Experimental: n = 6) and IV. Pharmacological inhibition of MnPO (MnPO Sham: n = 6; MnPO Experimental: n = 6). The animals of the first three protocols were anesthetized and subjected to saporin-anti-DβH nanoinjections for neuronal lesion (100 nL, 0.105 ng/nl) in experimental groups and Saporin nanoinjections (100 nL, 0.022 ng/nL) in sham groups for fictitious neuronal lesion, respectively, in the NTS, CVLM or simultaneously in the NTS and CVLM regions. After 20 days of recovery, the animals were anesthetized and instrumented to mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nervous activity (RSNA) recordings. Then, HH was performed by blood withdrawal until MAP reached approximately 60 mmHg. After 20 min of HH, sodium overload (3M NaCl, 1.8 mL/g, 90 seconds of infusion, i.v) was conducted. In another series of experiments, MnPO Sham and MnPO Experimental groups were anesthetized and instrumented for MAP, HR and RSNA recordings. Then, the animals were submitted to HH and HS infusion at the end of the hemorrhage. GABAergic agonist Muscimol (4 mM, 100 nL, MnPO Experimental group) or saline nanoinjections (0.15 M, 100 nL, MnPO Sham group) were performed in the MnPO after 10 min from the start of HH. HH-induced hypotension, bradycardia and renal sympathoinhibition in the animals of the A2 Sham, A1 Sham, A1 + A2 Sham and MnPO Sham groups. In the sham groups, HS infusion after HH reestablished MAP, HR, and did not alter the renal sympathoinhibition generated during hypovolemia. In the A2 Experimental and A1 Experimental groups, the specific lesion of A1 or A2 neurons did not alter the hypotension, bradycardia and renal sympathoinhibition caused during HH. In addition, the A1 or A2 neurons specific lesion did not alter the reestablishment of MAP, HR and the RSNA reduction after HS solution infusion. However, in the animals of the A1 + A2 experimental group, the simultaneous A1 and A2 neurons lesion did not alter the decrease in MAP and HR observed during HH, but abolished renal sympathoinhibition. In addition, simultaneous A1 and A2 neurons lesion abolished MAP restoration and ANSR reduction after HS infusion, while HR restoration was not modified. In the MnPO experimental animals, MnPO nucleus inhibition did not alter the decrease in MAP and HR observed during HH, but abolished renal sympathoinhibition. However, MnPO inhibition abolished the MAP restoration and promoted strong sympathetic activation in the renal bed after HS infusion, while HR restoration was not modified. These results suggest that the A1, A2 neuronal clusters and MnPO nucleus are part of the integration and transmission information circuits about changes in plasma osmolarity, participating in cardiovascular and autonomic recovery induced by sodium chloride overload in animals submitted to HH.
Os benefícios hemodinâmicos e cardiovasculares provenientes do uso de solução salina hipertônica (SH) no tratamento da hemorragia hipotensiva (HH) são relatados há vários anos. Recentes investigações mostraram a participação de regiões do sistema nervoso central (SNC), como os grupamentos neuronais A1 (localizado na região caudoventrolateral do bulbo; CVLM), A2 (localizado no núcleo do tracto solitário; NTS) e do núcleo Pré-óptico mediano (MnPO) nas respostas hemodinâmicas induzidas pela sobrecarga de cloreto de sódio em animais normovolêmicos. Entretanto, o papel das estruturas acima relacionadas na recuperação cardiovascular e nas alterações autonômicas induzidas pela administração de solução SH em animais submetidos à HH ainda não foi avaliado. Assim, o presente estudo buscou avaliar o envolvimento dos grupamentos neuronais A1, A2 e do núcleo MnPO nas respostas cardiovasculares e autonômicas promovidas pela infusão de solução SH em animais hipovolêmicos. Para isto, ratos Wistar (280-320 g) foram separados em quatro protocolos: I. Lesão do grupamento neuronal A2 (Controle A2: n=6; Experimental A2: n=6); II. Lesão do grupamento neuronal A1 (Controle A1: n=6; Experimental A1: n=6); III. Lesões concomitantes dos grupamentos neuronais A1 e A2 (Controle A1 + A2: n=6; Experimental A1 + A2: n=6) e IV. Inibição farmacológica do núcleo MnPO (Controle MnPO: n=6; Experimental MnPO: n=6). Os animais dos três primeiros protocolos foram anestesiados e submetidos a nanoinjeções de saporina-anti-DβH para lesão neuronal (100 nL, 0,105 ng/nL) nos grupos experimentais e Saporina (100 nL, 0,022 ng/nL) nos grupos controles para lesão neuronal fictícia, respectivamente, no NTS, na região CVLM ou conjuntamente no NTS e CVLM. Após 20 dias de recuperação, os animais foram novamente anestesiados e instrumentalizados para registro da pressão arterial média (PAM), frequência cardíaca (FC) e atividade nervosa simpática renal (ANSR). Em seguida, a HH foi realizada através da retirada de sangue até que a PAM atingisse aproximadamente 60 mmHg. Após 20 min de HH foi conduzida a sobrecarga de sódio (NaCl 3M, 1,8 mL/kg, 90 segundos de infusão, i.v). Em outra série de experimentos, os animais dos grupos controle MnPO e Experimental MnPO foram anestesiados e instrumentalizados para registro da PAM, FC, ANSR. Em seguida, foram submetidos à HH e a infusão de solução SH ao final da hemorragia. Nanoinjeções do agonista GABAérgico, muscimol (4 mM, 100 nL, grupo experimental MnPO) ou salina (0,15 M; 100 nL; grupo controle MnPO) foram realizadas no MnPO após 10 min do início da HH. A HH promoveu hipotensão, bradicardia e simpatoinibição no território renal nos animais dos grupos controle A2, controle A1, controle A1 + A2 e controle MnPO. Nos grupos controle, a infusão de solução SH após a HH reestabeleceu a PAM, FC e não alterou a simpatoinibição renal gerada durante a hipovolemia. Nos animais dos grupos experimental A2 e experimental A1, a lesão especifica dos neurônios A1 ou A2 não alterou a hipotensão, bradicardia e simpatoinibição provocados durante a HH. Em adição, a lesão especifica dos neurônios A1 ou A2 não alterou o reestabelecimento da PAM, FC e a queda da ANSR gerada após a infusão de solução SH. Entretanto, nos animais do grupo experimental A1 + A2, a lesão simultânea dos neurônios A1 e A2 não alterou a queda da PAM, da FC observada durante a HH, mas aboliu a simpatoinibição renal. Ademais, a lesão simultânea dos neurônios A1 e A2 aboliu a restauração da PAM e a redução da ANSR após a infusão de solução SH, enquanto a restauração da FC não foi modificada. Nos animais do grupo experimental MnPO, a inibição do MnPO não alterou a queda da PAM e da FC observadas durante a HH, entretanto aboliu a simpatoinibição renal. Porém, a inibição do núcleo MnPO aboliu a restauração da PAM e promoveu forte simpatoexcitação no leito renal após a infusão de solução SH, enquanto a restauração da FC não foi modificada. Esses resultados sugerem que os neurônios dos grupamentos A1, A2 e o núcleo MnPO fazem parte dos circuitos de integração e transmissão de informações a respeito de mudanças na osmolaridade plasmática, participando da recuperação cardiovascular e autonômica induzida pela sobrecarga de cloreto de sódio em animais submetidos à HH.

碩士
國防醫學院
生理學研究所
87
We examined the involvement of caudal ventrolateral medulla(CVLM) in vasomotor and respiratory control. Experiment were carried out in cats under a-chloralose/urethane anesthesia under artificial ventilation to maintain the end-expiratory CO2 at 3.5~4.5 %. Phrenic nerve activity(PNA) was recorded to monitor the changes of respiratory frequency(Rf) and inspiratory depth. Microinjection of sodium glutamate(Glu) into CVLM decreased systemic arterial blood pressure(SAP) and altered phrenic nerve activities(PNA). Among 143 depressor sites, 55 %(78/143) increased Rf, while 72 % altered inspiratory depth(36 % increased and 36 % decreased). A small but significant positive correlation was observed between the magnitudes of depressor responses and inhibition of inspiratory depth neurons. Furthermore, microinjections of acetylcholine(ACh) mimicked the Glu-induced depressor responses. However, ACh did not alter Rf, but still reduced inspiratory depth. Our findings suggest that Rf-regulating and depressor neurons are two separate neuronal population, coexisting in CVLM. The inspiratory depth inhibitory and depressor neurons, in contrast, could have stronger correlation. Inhibitory amino acid: g-aminobutyric acid(GABA) and Glycine(Gly) in the depressor/inspiratory depth-inhibition sites increased SAP while decreased inspiratory depth. GABA and Gly effects on Rf are different. Gly increased Rf, while GABA had no effect. After kainic acid lesion of Rf-increased neurons in the CVLM, the generation of PNA is severely disturbed.

博士
國立陽明大學
藥理學研究所
100
Intoxication from the psychostimulant methamphetamine (METH) because of cardiovascular collapse is a common cause of death within the abuse population. The demonstration that successful resuscitation of an arrested heart depends on maintained functionality of the rostral ventrolateral medulla (RVLM), which is responsible for the maintenance of stable blood pressure, suggests that failure of brain stem cardiovascular regulation, rather than the heart, holds the key to cardiovascular collapse. The present study evaluated the hypothesis that METH effects acute cardiovascular depression by dampening the functional integrity of baroreflex via an action on RVLM. Moreover, this study further tested the hypothesis that cessation of brain stem cardiovascular regulation because of a loss of functionality in RVLM mediated by bioenergetics failure and oxidative stress underlies the cardiovascular collapse elicited by acute intoxication of METH. The distribution of METH in brain and heart on intravenous administration in male Sprague-Dawley rats, and the resultant changes in arterial pressure (AP), heart rate (HR) and indices for baroreflex-mediated sympathetic vasomotor tone and cardiac responses were evaluated, alongside survival rate and time. Intravenous administration of METH (12 or 24 mg/kg) resulted in a time-dependent and dose-dependent distribution of the psychostimulant in brain and heart. The distribution of METH to neural substrates associated with brain stem cardiovascular regulation was significantly larger than brain targets for its neurological and psychological effects; the concentration of METH in cardiac tissues was the lowest among all tissues studied. In animals that succumbed to METH, the baroreflex-mediated sympathetic vasomotor tone and cardiac response were defunct, concomitant with cessation of AP and HR. On the other hand, although depressed, those two indices in animals that survived were maintained, alongside sustainable AP and HR. Linear regression analysis further revealed that the degree of dampening of RVLM cardiovascular regulation was positively and significantly correlated with the concentration of METH in the key neural substrate involved in this homeostatic mechanism. High doses of METH induced significant mortality within 20 min that paralleled concomitant the collapse of AP or HR and loss of functionality in RVLM. There were concurrent increases in the concentration of METH in serum and ventrolateral medulla, along with tissue anoxia, cessation of microvascular perfusion in RVLM. Furthermore, mitochondrial respiratory chain enzyme activity or electron transport capacity and ATP production in RVLM were reduced, and mitochondria-derived superoxide anion level was augmented. All those detrimental physiological and biochemical events were reversed on microinjection into RVLM of a mobile electron carrier in the mitochondrial respiratory chain, coenzyme Q10; a superoxide dismutase mimetics, 4-hydroxy-2,2, 6,6-tetramethylpiperidine-1-oxyl; or a mitochondria-targeted antioxidant and superoxide anion scavenger, Mito-TEMPO. In animals that survived, toxic doses of METH induced apoptotic cell death in RVLM. The physiological changes and ATP loss in RVLM were restored by an apoptosome inhibitor, 4-chloro-2-[3-(3-trifluoromethyl -phenyl)-ureido] benzoic acid (NS3694). On the other hand, necrotic cell death in RVLM was found in animals that deceased following METH administration. Comparably, the physiological changes and ATP loss in RVLM of these animals were effectively reversed by an oxidative stress-induced necrotic cell death inhibitor, (2-[1H-indol-3-yl]-3-pentylamino-maleimide (IM-54, 3 pmol), but not by NS3694. In conclusion, on intravenous administration, METH exhibits a preferential distribution to brain stem nuclei that are associated with cardiovascular regulation. The concentration of METH in those brain stem sites dictates the extent that baroreflex-mediated sympathetic vasomotor tone and cardiac responses are compromised, which in turn determines survival or fatality because of cardiovascular collapse. Furthermore, METH-induced apoptotic or necrotic mechanisms in RVLM may differentially play determinant roles in the survival of animals. This study found that sustained anoxia and cessation of local blood flow that leads to bioenergetics failure and oxidative stress because of mitochondrial dysfunction, leading to acute necrotic cell death in RVLM underpins cardiovascular collapse elicited by lethal doses of METH.

博士
國立中山大學
生物科學系研究所
91
Dysfunction of Mitochondrial Respiratory Chain in Rostral Ventrolateral Medulla During Experimental Endotoxemia Sepsis is a complex pathophysiologic state resulting from an exaggerated whole-body inflammatory response to infection or injury. Metabolic disturbances, abnormal regulation of blood flow and diminished utilization of oxygen at the cellular level may account for tissue damage and lead to multiple organ failure and death. As the primary site of cellular energy generation is the mitochondrion, it presents itself as an important target for the septic cascade. In this regard, the notion that bioenergetic failure due to mitochondrial dysfunction contributes to organ failure during sepsis has received attention. We established the low frequency fluctuations in the systemic arterial pressure signals are related to the sympathetic neurogenic vasomotor tone, and reflect the functional integrity of the brain stem. Their origin is subsequently traced to the premotor sympathetic neurons at the rostral ventrolateral medulla (RVLM), whose neuronal activity is intimately related to the “life-and-death” process. Based on a rat model of experimental endotoxemia that provides continuous information on changes in neuronal activity in the RVLM, the present study was undertaken to evaluate whether changes in mitochondrial respiratory functions are associated with death arising from sepsis. We also evaluated the efficacy of a new water-soluble coenzyme Q10 (CoQ10, ubiquinone) formula in the protection against fatality during endotoxemia by microinjection into bilateral RVLM. Dysfunction of Mitochondrial Respiratory Chain in Rostral Ventrolateral Medulla During Experimental Endotoxemia in the Rat We investigated the functional changes in mitochondrial respiratory chain at the RVLM in an experimental model of endotoxemia that mimics systemic inflammatory response syndrome. Experiments were carried out in adult male Sprague-Dawley rats that were maintained under propofol anesthesia. Intravenous administration of E. coli lipopolysaccharide (LPS; 30 mg/kg) induced progressive hypotension, with death ensued within 4 hours. The sequence of cardiovascular events during this LPS-induced endotoxemia can be divided into a reduction (Phase I), followed by an augmentation (Phase II; “pro-life” phase) and a secondary decrease (Phase III; “pro-death” phase) in the power density of the vasomotor components (0-0.8 Hz) of systemic arterial pressure (SAP) signals. Enzyme assay revealed significant decrease of the activity of NADH cytochrome c reductase (Complex I+III) and cytochrome c oxidase (Complex IV) in the RVLM during all 3 phases of endotoxemia. On the other hand, the activity of succinate cytochrome c reductase (Complex II+III) remained unaltered. Neuroprotective Effects of Coenzyme Q10 at Rostral ventrolateral Medulla Against Fatality During Experimental Endotoxemia in the Rat CoQ10 is a highly mobile electron carrier in the mitochondrial respiratory chain that also acts as an antioxidant. We evaluated the neuroprotective efficacy of CoQ10 against fatality in an experimental model of endotoxemia, using a novel water-soluble formulation of this quinone derivative. In Sprague-Dawley rats maintained under propofol anesthesia, intravenous administration of E. coli LPS (30 mg/kg) induced experimental endotoxemia. Pretreatment by microinjection bilaterally of CoQ10 (1 or 2 mg) into RVLM significantly diminished mortality, prolonged survival time, and reduced the slope or magnitude of the LPS-induced hypotension. CoQ10 pretreatment also significantly prolonged the duration of Phase II endotoxemia and augmented the total power density of the vasomotor components of SAP signals in Phase II endotoxemia. The increase in superoxide anion production induced by LPS at the RVLM during Phases II and III endotoxemia was also significantly blunted. Conclusion The present study revealed that selective dysfunction of respiratory enzyme Complexes I and IV in the mitochondrial respiratory chain at the RVLM is closely associated with fatal endotoxemia. CoQ10 provides neuroprotection against fatality during endotoxemia by acting on the RVLM. We further found that a reduction in superoxide anion produced during endotoxemia at the RVLM may be one of the mechanisms that underlie the elicited neuroprotection of CoQ10. These findings therefore open a new direction for future development of therapeutic strategy in this critical, complicated and highly fatal condition known as sepsis.

The rostral ventrolateral medulla (RVL) contains a population of bulbospinal neurons which are critically involved in the tonic and reflex control of blood pressure. They project to the intermediolateral cell column of the thoracolumbar spinal cord and excite sympathetic preganglionic neurons. The RVL region also contains the C1 adrenergic group of neurons, some of which are also bulbospinal RVL vasomotor neurons. The objective of this study was to investigate RVL neurons at the cellular and molecular level, to gain a better understanding of the properties of this heterogeneous population of neurons. Two main approaches were used to achieve this. The first studied the expression of several genes in RVL micropunches and single acutely dissociated RVL neurons using the reverse transcription-polymerase chain reaction (RT-PCR). The second utilised antisense oligodeoxyribonucleotides (aODNs) to reduce expression of two adrenaline-synthesising genes, tyrosine hydroxylase (TH) or phenylethanolamine N-methyltransferase (PNMT), in C1 adrenergic neurons in the RVL. Semi-nested RT-PCR analysis of tissue micropunched from the RVL region of adult rats established the expression of mRNA transcripts for glyceraldehydes 3-phosphate dehydrogenase (GAPDH), neuron-specific enolase (NSE), TH, PNMT, the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), noradrenaline transporter (NET), glycine transporter 2 (GLYT2), neuronal glutamate transporter (EAACI) and glial glutamate transporter (GLT). The expression of mRNA for glutamic acid decarboxylase (GAD67) and the gamma-amino butyric acid (GABA) transporter (GAT-1) was also detected in rat brainstem. The micropunch technique combined with RT-PCR offers a simple non-radioactive way to identify genes being expressed in the RVL and other medullary regions. Single neurons were acutely dissociated from the RVL of 13 - 2l day old rats, and classified as spinally projecting or non-spinal by the presence or absence of retrogradely-transported fluorescent beads injected into the upper thoracic segments. Dissociated bulbospinal RVL neurons did not exhibit any spontaneous firing, and there was no evidence of any pacemaker-like properties. A fast-activating (spike-like) inward current could be elicited by depolarising voltage steps, and could be abolished by application of tetrodotoxin. Extracellular application of either kainic acid or L-glutamic acid onto RVL cells under voltage-clamp conditions elicited dose-dependent inward currents. Individual neurons were collected by aspiration into a glass micropipette and the cell contents analysed by RT-PCR. Detection of either GAPDH or NSE mRNA was used as the criterion for selecting cells for further analysis, and 80% of the neuron samples tested expressed one of these genes. A subpopulation of spinally-projecting neurons expressed PNMT or TH (50%), indicating that they were catecholaminergic. Bulbospinal RVL neurons were also found to express mRNA for the MR (45% of those tested), the GR (5%), NET (10%), and EAAC1 (58%). GLT, GAD67 and GAT-1 were not expressed in any bulbospinal neurons, but they were detected in 5 - l0% of non-spinal neurons tested. Expression of mRNA for MR, EAAC1 and NET was also observed in subpopulations of PNMT-positive and -negative bulbospinal neurons. Expression of NET protein, assessed by immunohistochemistry, was found to be similar to NET mRNA expression in C1 adrenergic neurons in the RVL. These results indicate that single-cell RT-PCR is a powerful method for elucidating the functional characteristics of a defined neuronal population, and that it is possible to perform whole-cell patch-clamp recording prior to RT-PCR analysis, allowing linkage of the molecular analysis of mRNA expression to the electrophysiological properties of these neurons. The method is very sensitive, enabling low copy-number mRNA transcripts to be detected, and can be used to provide key information about blood-pressure regulation at the molecular and cellular level. The final part of this study used aODNs to attempt to knockdown expression of TH or PNMT in C1 neurons in the RVL. The aODNs were either injected or infused unilaterally into the RVL of adult rats in vivo, and 4 - 14 days later the animals were sacrificed and the expression of TH or PNMT protein assessed using immunohistochemistry. Immunoreactivity for TH was unchanged by injection or infusion of an aODN targeting TH mRNA. Immunoreactivity for PNMT was decreased in 2 out of l0 infusion experiments, but this was achieved using an aODN with one base pair mismatch to the PNMT mRNA sequence, and could not be repeated with aODNs of the correct sequence. This study was the first attempt to target the PNMT gene with aODNs, and although preliminary in nature, it provided some useful information for future studies of this system. Antisense ODN suppression of genes in the RVL may prove to be an interesting future avenue for investigating the functional significance of specific genes being expressed in the RVL region.

博士
國立中山大學
生物醫學研究所
99
An individual who has sustained either irreversible cessation of circulatory and respiratory functions, or irreversible cessation of all functions of the entire brain, including the brain stem is dead. Brain death is currently the legal definition of death in many countries. Many people confuse brain death with vegetative states. Patients in a vegetative state are unaware of themselves or their environment. Both patients with brain death and those in a vegetative state are unconscious following severe brain injury. Unlike the brain death, vegetative patient’s vital vegetative functions, such as cardiac action, respiration, and maintenance of blood pressure are preserved. The rostral ventrolateral medulla (RVLM) is the origin of a “life-and-death” signal identified from systemic arterial blood pressure spectrum and intimately related to brain death. Based on the animal models of brain death, the observations that the power density of the vasomotor components of SAP signals undergoes both augmentation and reduction during the progression towards death strongly suggest that both ‘‘pro-life’’ and ‘‘pro-death’’ programs are present in the RVLM. A number of those ‘‘pro-life’’ and ‘‘pro-death’’ programs in the RVLM has now been identified along with their cellular and molecular mechanisms. As the neural substrate that is intimately related to brain death, one unresolved question is whether the proteome expressed in RVLM is unique. To address the issue, we used the cerebral cortex, which is defunct under persistent vegetative state for comparison. 2-DE electrophoresis, MALTI-TOF MS and peptide mass fingerprinting were used for investigation the proteomic difference between the rat RVLM and cerebral cortex. Quantitative analysis on silver-stained 2-DE electrophoresis gels revealed highly comparable distribution patterns of these protein spots for both brain regions, with 85.9 ± 2.3 % of protein spots from RVLM matched those from cerebral cortex. According to the protein function, these proteins were classed into binding activity, chaperone, antioxidant, oxidoreductase, ubiquitin- proteasome system, cell cycle, catalytic activity, glycolysis, tricarboxylic acid cycle, electron transport chain, endocytosis and exocytosis, structural molecular function, apoptosis, transport, differentiation and neurogenesis, protein biosynthesis, cell junction, and others. We found that a group of antioxidant proteins, including members of the peroxiredoxin (Prx) family (Prx-1, Prx-2, Prx-5, and Prx-6), thioredoxin and mitochondrial manganese superoxide dismutase exhibited significantly higher protein and mRNA expression levels in RVLM when compared to cerebral cortex. Tissue oxygen, ATP contents and ATP synthase subunits alpha and beta in RVLM were also significantly elevated. On the other hand, protein and mRNA levels of members of the ubiquitin-proteasome system, including proteasome subunit alpha type-1, ubiquitin, uniquitin-conjugating enzyme E2 N, ubiquitin carboxyl-terminal hydrolase isozyme L1 and L3, were comparable in both brain regions. The presence of higher levels of tissue oxygen and ATP synthase subunits in RVLM, leading to augmented ATP production, provides a cellular safeguard mechanism to reduce the possibility of irreversible reduction in intracellular ATP contents that precipitate brain death. By manifesting an augmented tissue oxygen and metabolic energy production, RVLM is more prone to oxidative stress. We conclude that a significantly elevated level of antioxidant proteins and mRNA in RVLM is consistent with the exhibition of higher tissue oxygen tension and metabolic energy production in this neural substrate, which together constitute a safeguard mechanism against brain death.

博士
國立成功大學
基礎醫學研究所
102
Regulation of the arterial blood pressure is a complex and delicate physiological process. Vascular dysfunction, alterations in the humeral factors, including the circulatory metabolites, angiotensin II (Ang II), superoxide anion, nitric oxide and carbon monoxide, are all engaged in pathophysiology of the high blood pressure. At the same time, perturbation of the central cardiovascular regulatory machineries also contributes to pathogenesis of hypertension. It is well established that overexcitation of the premotor neurons in the rostral ventrolateral medulla (RVLM) results in sympathoexcitation and the increase in arterial blood pressure. Previous studies have shown that oxidative stress of an enhanced production of the superoxide anion (O2.-) and/or hydrogen peroxide (H2O2) in the RVLM plays a pivotal role in manifestation of hypertension of the spontaneously hypertensive rats (SHR). The endoplasmic reticulum (ER) is the first compartment of the secretory pathways to rescue unfolded proteins in cells. When misfolded or unfolded proteins accumulate in the ER, the cells activate a self-protective mechanism, termed the ER stress. Prolonged ER stress has been proposed to be involved in the neurodegenerative diseases. In this study, we hypothesized that ER stress may underpin the cellular events leading to the manifestation of redox-associated neurogenic hypertension. We found that in comparison with the normotensive Wistar-Kyoto (WKY) rats, the expression of two major marker proteins of ER stress, glucose-regulated protein 78 (GRP78) and the phosphorylated eukaryotic initiation factor 2α (eIF2α), at RVLM was significantly greater in the SHR. Intraperitoneal or intracerebroventricular infusion of Ang II or direct microinjection of a ER stress inducer, tunicamycin, promoted ER stress and increased the systemic arterial pressure in the normotensive WKY rats. Protection of the RVLM cells from undertaking ER stress by microinjection bilaterally into the RVLM of salubrinal, on the other hand, caused a significant decrease in systolic arterial pressure of SHR, alongside a concomitant suppression of the increased GRP78 expression and downregulation of eIF2α phosphorylation. We further found that ER stress induced activation of autophagy in RVLM, leading to hypertension in the SHR. Finally, we identified that oxidative stress activated the ER stress through the activation of PI3K/Akt signaling and this redox-sensitive activation of PI3K/Akt signaling was engaged in the neurogenic hypertension. Collectively these results suggest that exaggerated ER stress may be an important factor in the manifestation of neurogenic hypertension via redox-sensitive activation of PI3K/Akt pathway and autophagy in the RVLM.

碩士
慈濟大學
藥理暨毒理學碩士班/博士班
100
Ethanol (alcohol) intake affects cardiovascular system. The precise mechanisms underlying ethanol regulation of cardiovascular function remains unclear. Alterations of central sympathetic activity has been suggested to participate in ethanol-induced changes in blood pressure. Emerging evidence indicates that glutamate system may play a crucial role in the regulation of several neurobiological effects of ethanol. Glutamate is the major excitatory neurotransmitter mediating the activity of pre-sympathetic neurons in the rostral ventrolateral medulla (RVLM), key neurons involved in the regulation of sympathetic activity and cardiovascular functions in the central nervous system. Glutamate-mediated excitability in RVLM neurons has been shown to be different between the spontaneously hypertensive rats (SHR) and the Wistar-Kyoto normotensive (WKY) rats. The present study examined the role of glutamate system in the RVLM in ethanol regulation of cardiovascular functions in the SHR, WKY and SD rats. Intraperitoneal administration of ethanol caused a significant decrease in blood pressure in SHRs and WKY and SD rats. However, ethanol-induced depressor responses were more obvious in SHRs and SD rats than those in WKY rats. Intraperitoneal injection of highs dose ( 3.2g/kg) of ethanol caused a significant increase in the levels of glutamate in the RVLM in the SHR and SD rats, but no significant change in the WKY rats. Bilateral microinjection of MPEP, an antagonist for metabotropic glutamate receptor subtype 5, into the RVLM 5 min after administration of ethanol had little effects on ethanol-induced depressor effects in SHR and WKY rats. However, post-treatment with AP-5, Memantine, Ketamine, NMDA glutamate receptor antagonists, significant reduced ethanol-induced depressor effects in SHR rats. The results showed that different strains of rats may have different sensitivity to ethanol effects, and NMDA receptors in the RVLM may participate in ethanol regulation of cardiovascular function.

碩士
國防醫學院
藥理學研究所
86
The rostral ventrolateral medulla (RVLM) and nucleus tractus solitarii (NTS) are two important brain stem nuclei in central cardiovascular regulation. The neurons in RVLM project to presympathetic neurons of spinal cord and maintain the sympathetic nerve activity, whereas NTS integrates peripheral messages and modulates the reflex activity of cardiovascular effects.Nitric oxide (NO) is a remarkable regulation molecule which plays an important role in multiple physiological functions. In this study, we examined the cardiovascular effects of NO in RVLM of rats, and the role of NO in modulating the baroreflex in NTS of hypertensive rats. The cardiovascular effects of NO in RVLM of Sprague-Dawley rats (250-350 g) were investigated. Drugs were applied to the RVLM or NTS by sterotaxic microinjection technique and their cardiovascular effects were evaluated. The role of NO in baroreflex response was evaluated in SD and 2K1C rats, which were established by clamping the left renal arte ry of SD rats (180-200 g) with a silver clip. The induced elevation in blood pressure and the decrease in heart rate, caused by phenylephrine injections, were used to calculate the index of baroreflex sensitivity. We observed that unilateral microinjection of L-arginine (90 nl) into the RVLM produced obvious depressor effects. However, D-arginine and L-NMMA (a NO synthase inhibitor) had no significant cardiovascular effects. Pretreatment of L-arginine attenuated the pressor effect of L-glutamate, while pretreatment of D-arginine or L-NMMA did not. Microinjection of L-arginine reduced the pressor effect of NMDA, but not that of AMPA (non-NMDA receptor agonist). In the RVLM, the depressor effect of GABA was reduced by pretreatment of L-arginine, but not by D-arginine or L-NMMA. The depressor effect of baclofen (GABAB receptor antagonist), but not that of muscimol (GABAA receptor antagonist), was attenuated by L-arginine. 2K1C rats had an elevated plasma NO level at 1 week (1W) and returned to normal at 4 weeks (4W) after operation, compared to values of SD rats. Significant hypertension was observed 4W but not 1W after operation. The baroreflex sensitivities of 2K1C rats (1W, 4W) were lower than that of SD rats. Microinjection of ADMA (an endogeneous NOS inhibitor) attenuated the baroreflex sensitivity in S D rats, but not the sensitivity of 2K1C rat (4W). In conclusion, our data suggest that NO may produce direct depressor effect in RVLM, and may be involved in cardiovascular regulation by modulating the effects of glutamate and GABA. Secondly, the loss of NO-dependent component of baroreflex may be the cause of reduced barorelfex sensitivity in 2K1C rats. The results show that NO plays an important role in the cardiovascular regulation of RVLM and NTS.

碩士
中原大學
醫學工程學系
87
The aim of the present study is to examine the relationship between the lateral tegmental field (FTL), a depressor area, the gigantocellular tegmental field (FTG), a pressor area, and the depressor caudal ventrolateral medulla (CVLM) of the brain stem; and the relationship between FTG and two cardioinhibitory areas, i.e., the ambiguus nucleus (AN) and the dorsal motor nucleus of vagus (DMV). Fast fourier transform (FFT) was used to analyze the basal vertebral nerve activity (VNA) and the systemic arterial pressure (SAP) in frequency domain. Sixty five cats were anesthetized intraperitoneally with chloralose (40mg/kg) and urethane (400mg/kg). Systemic arterial pressure (SAP), heart rate (HR) and the sympathetic vertebral nerve activity (VNA) were recorded. The correct location of FTL, FTG, CVLM, AN or DMV was determined first by electrical stimulation ( 80 Hz, 0.5 ms, 50 -100 uA ) then followed by microinjection of sodium glutamate (Glu, 0.25M, 70nl). It was found that the depressor responses caused by the FTL stimulation were greatly reduced after lesioning the CVLM by microinjection of kainic acid (KA, 24mM, 150nl) ipsilateral to the side of the FTL stimulation. Data suggest that the FTL-depressor responses probably are mediated through excitation of the depressor neurons in CVLM. The FTG pressor response were reduced after the CVLM was lesioned. Data suggest that the FTG-pressor response probably are mediated through inhibition of the neurons in CVLM. Lesioning the DMV or AN by KA reduced the FTG-induced bradycardiac responses. The reduction after the AN lesion ,however, was more pronounced than that after DMV lesion. Data suggest that part of the FTG bradycardiac action is mediated through activation of the AN or DMV.

碩士
國立中山大學
生物科學系研究所
91
Heat shock proteins (HSPs) are abundantly produced in cells that are under stress or injury by acting as a chaperone or promoting folding, unfolding, packing, degradation or denaturing of proteins or peptides. This study evaluated the role of HSP60, HSP70 or HSP90 in the rostral ventrolateral medulla (RVLM), in experimental endotoxemia in the rat. Adult, male Sprague-Dawley rats maintained by i.v. infusion propofol (25 mg/kg/h) were used. During experimental endotoxemia induced by intravenous administration of E. coli lipopolysaccharide (LPS, 30 mg/kg; serotype 0111:B4), the power density of the vasomotor component of systemic arterial pressure (SAP) spectrum underwent a decrease (Phase I), followed by an increase (Phase II; “pro-life”) and a secondary decrease (Phase III; “pro-death”). Western blot analysis revealed that HSP60 expression in the RVLM was significantly increased during Phase II and Phase III endotoxemia; and HSP70 expression was maximally increased during Phase II. HSP90 protein expression in the RVLM was not significantly changed during endotoxemia. We further studied the role of HSP60, HSP70 or HSP90 at the RVLM in experimental endotoxemia by pretreating animals with bilaterally microinjection of an anti-HSP serum (HSPAb, 1:20), normal mouse serum, antisense oligodeoxynucleotide (hsp AODN, 50 pmol), sense oligodeoxynucleotide (hsp SODN) or scrambled AODN (hsp SC). Pretreatment with HSP60Ab or hsp60 AODN resulted in significantly higher mortality, shorter survival time and shorter Phase II duration. In addition, the augmented power density of the vasomotor component of SAP signals during Phase II endotoxemia was significantly reduced. Even more detrimental effects were obtained on local application of HSP70Ab or hsp70 AODN into the RVLM. Pretreatment with HSP90Ab or hsp90 AODN was ineffective. We conclude that the expression of HSP60 and HSP70 in the RVLM may play a “pro-life” role in fatal experimental endotoxemia; and HSP90 may not be involved.

碩士
國立中山大學
生物科學系研究所
91
We investigated the role of muscarinic receptor subtypes at the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic neurogenic vasomotor tone, in mevinphos (Mev) intoxication. Adult Sprague-Dawley rats anesthetized by pentobarbital (45 mg/kg) and maintained by propofol (30 mg/kg/h) were used. Co-microinjection bilaterally of Mev (10 nmol) and artificial cerebrospinal fluid (aCSF) into the RVLM resulted in an increase (Phase I) followed by a decrease (Phase II) in the power density of the vasomotor components of systemic arterial pressure spectrum, our experimental index for sympathetic vasomotor tone. These changes in sympathetic vasomotor outflow in both phases of Mev intoxication were significantly and dose-dependently reduced on co-microinjection of Mev and the M2 subtype of muscarinic receptor (M2R) antagonist methoctramine (0.5 or 1 nmol) or M4R antagonist tropicamide (0.5 or 1 nmol). On the other hand, the M1R antagonist pirenzepine (0.5 or 1 nmol) or M3R antagonist 4-DAMP (0.5 or 1 nmol) was ineffective. Western blot analysis further revealed that the increase in NOS I protein levels at the RVLM during Phase I Mev intoxication or the augmented level of NOS II during both phases were significantly blunted on co-microinjection bilaterally of Mev and methoctramine (1 nmol) or tropicamide (1 nmol) into the RVLM. Pirenzepine (1 nmol) or 4-DMAP (1 nmol) was again ineffective. We conclude that both M2R and M4R subtypes in the RVLM may be involved in Mev intoxication. Whereas the prevalence of NOS I over NOS II at the RVLM during Phase I results in sympathoexcitation, sympathoinhibition induced by NO from NOS II in the RVLM is primarily involved in Phase II Mev intoxication.

碩士
國立臺灣大學
動物學研究所
85
Neural factor is one of the main cause of hypertension. It is believed that a enhanced sympathetic activity plays a critical role in the development and the maintenance of essential hypertension. This thesis studied the possible involvement of the parasympathetic system. Stroke-prone spontaneously hypertensive rat (SP) and spontaneously hypertensive rat (SHR), two of the most widely used animal models of essential hypertension, were used as hypertensive rat. Wistar Kyoto rat (WKY) and Wistar rat were used as normotensive controls. Rats were anesthetized with urethane and α-chloralose. Microinjections of glutamate solution (110 pmole) in the ventrolateral region of the medulla were used to probe the cardiac vagal area. Heart rate, blood pressure and diaphragm electromyogram responses were recorded. The maximal responses of heart rate decreases were larger in Wistar than either WKY or SP. With percentile scale, however, there were no differences among the four stains. Hypertensive rats had larger depressor responses. The results support that hypertensive rats has an enhanced sympathetic control in the medulla, whereas their parasympathetic cardioinhibitory control is not changed. In the medulla, heart rate, blood pressure, and respiration points were interrelated. There was no difference in the distribution of all three types of control points.

碩士
國立臺灣師範大學
生物研究所
88
The purpose of the present study was to examine the existence of the neural pathway projecting from the rostral ventrolateral medulla (rVLM) to the nucleus ambiguus (NA) by a technique of antidromic identification. Adult cats were anesthetized with pentobarbital sodium (30 mg/kg, i.p.). Bilateral vagotomy, paralyzation, and artificial ventilation were performed. Animals were maintained at normocapnia or hypercapnia in hyperoxia. Single unit activity in the rVLM was explored extracellularly. Electrical current (0.3 ms pulse duration, 1 Hz, and a variety of current intensity) was delivered to the NA once a single rVLM unit was verified by displaying the same amplitude and shape of the action potential on the oscilloscope. Of the 70 neurons examined, 49 neurons showed respiratory-related discharge pattern (10% inspiratory, 27% expiratory, 12% inspiratory-expiratory, 12% expiratory-inspiratory, and 39% tonic). Of the 49 recording sites tested, 1 unit could be activated antidromically by the delivery of current to the NA. This antidromic spike followed a 50 Hz repetition rate and could be collided with an orthodromic spike. Another unit could be activated orthodromically from the NA. In addition, there were evoked potentials in 15 recording sites. Moreover, 6 of the 7 respiratory-related neurons examined showed a tendency to increase their discharge rate in response to hypercapnia in hyperoxia. The present results demonstrate that the neurons in the rVLM may send axonal projections to the NA. These neural pathways may modulate the respiratory-related neuron activities in the NA which, in turn, influence the respiratory-related activities of the peripheral nerves originating from the NA.

碩士
國立臺灣師範大學
生物學系
84
The purpose of the present study was to evaluate the effect of the rostralventrolateral medulla (rVLM) on respiratory- related activity of the recurrentlaryngeal nerve (RLN). The animal was decerebrated, vagatomized paralyzed and ventilated artificially. Activities of the phrenic (PNA) and recurrent laryngeal nerve (RLNA) were monitored at normocapnia or hypercapnia both in hyperoxia. The rVLM was activated by electrical current (12.5 to 50 mA, 80 Hz, 0.5 ms) or glutamate (50 mM, 30-60 nl). Changes of PNA and RLNA are observed. In response to current of 12.5, 25 and 50 mA delivered to the rVLM, PNA were reduced to 93.3%, 74.9% (p<0.01) and 50.3% (p<0.01) of control respectively. Meanwhile, inspiratory RLNA (iRLNA) were reduced to 85.1% (p<0.05), 71.6% (p<0.01) and 54.7% (p<0.01) of control and expiratory RLNA (eRLNA) are reduced to 84.4% (p<0.05), 75.5% (p<0.01) and 63.6% (p<0.01) of control. With glutamate microinjection into the rVLMs, PNA, inspiratory and expiratory RLNA were decreased to 65.4% (p<0.01), 75.2% (p<0.01) and 53.3% (p<0.01) of control. This inhibition of the rVLM upon PNA and RLNA was attenuated by high level of FETCO2. Hence, at mild hypercapnia ( FETCO2=0.06), 25 mA delivery to the rVLM produced a decrease of PNA to 83.4% (p<0.05), iRLNA to 78.5% (p<0.01) and eRLNA to 62.8% of control. 50 mA delivery to the rVLM produced a decrease of PNA to 56.3% (p<0.01), iRLNA to 53.5% (p<0.01) and eRLNA to 43.9% (p<0.01) of control. When FETCO2 was further raised to 0.09, current of 25 mA excited to the rVLM could produce a decrease of PNA to 89.5% (p<0.05), iRLNA to 87.2% and eRLNA to 77.4% (p<0.05) of control. 50 mA delivery to the rVLM produced a decrease of PNA to 65.5% (p<0.01), iRLNA to 59.2% (p<0.01) and eRLNA to 44.3% (p<0.01) of control. These results suggest that the rVLM may play an important role in the modulation mechanism of upper airway resistance.

碩士
慈濟大學
生理暨解剖醫學碩士班
102
Cocaine- and amphetamine-regulated transcript peptide (CARTp) is widely expressed in the rat central nervous system, notably in areas involved in control of autonomic and neuroendocrine functions. CARTp-immunoreactivity was detected in the neurons and nerve terminals of the rostral ventrolateral medulla (RVLM) which is critical to the tonic and reflexive regulation of arterial blood pressure. In urethane anesthetized Sprague-Dawley (SD) rats, central administration of CARTp can induce significant increases in blood pressure, which accompanied an increase of c-Fos-like immunoreactivity in the RVLM. Our previous studies showed that bilateral microinjection of CARTp (6, 30 or 60 pmol) into the RVLM increased the mean arterial pressure (MAP) in SD rats. These evidences implicated that the RVLM may be the central target structure for CARTp participating in the cardiovascular regulation. In this study, we found that bilateral microinjection of CARTp 30 pmol into the RVLM increased the MAP in both spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats, but significant pressor effect of low dose CARTp (6 pmol) was observed only in SHRs. Bilateral intra-RVLM injections of CARTp antibody (1:5000) decreased the MAP in SHRs but not WKY rats. Concomitant administration of CARTp (30 pmol) and CARTp antibody (1:5000) into the RVLM did not cause changes of MAP in both SHRs and WKY rats. Using immunohistochemistry study, the number of CARTp-immunoreactive neurons in the RVLM was counted in WKY rats and SHRs. SHRs presented a greater number of CARTp neurons in the RVLM than WKY rats. Our data suggest that CARTp is involved in the pathophysiology and maintenance of high blood pressure in the SHR.

碩士
國立臺灣師範大學
生物研究所
88
Abstract The aim of the present study was to examine the projections from the rostral ventrolateral medulla (RVLM) of the rat to the nucleus ambiguous (NA), and to other respiratory and cardiovascular related nuclei in the medulla and spinal cord by tract-tracing methods. Two experiments were performed. In the first experiment, retrograde tracer WGA- HRP was injected into the recurrent laryngeal nerves of three rats. 3 days lateral, HRP-labeled neurons were detected mainly in the ipsilateral NA and its neighborhood. These cells distributed along the longitudinal axis of NA with two major peaks. Nine rats were used in the second anterograde tracing experiment. First, the RVLM were activated with microinjection of glutamate solution（50mM）to locate an area which produced significant decreases in diaphragm activity and increase in blood pressure. Then BDA, an anterograde tracer, was injected into the same area. 10 to 14 days later, fiber terminals radiated from these RVLM injection sites were found in the bilateral NA, the hypoglossal nucleus, the solitary tract nucleus, the dorsal motor nucleus of the vagus, the phrenic motor nucleus (PMN) in the cervical spinal cord, and the intermediolateral cell column (IML) in the thoracic spinal cord, all these nuclei related to respiratory and cardiovascular function. The correlation between terminal density in the PMN and IML from the RVLM versus glutamate- induced functional changes, i.e., diaphragm activity depression (for PMN) or blood pressure increase (for IML), were examined with linear regression statistics. Significant correlation was found between the terminal density in the PMN and the suppression of diaphragm muscle EMG. The correlation between terminal density in the IML and blood pressure increase did not reach statistically significant level. Findings of the present study suggest that RVLM play an important role in modulating or controlling respiratory and cardiovascular functions by direct projections to respiratory and cardiovascular related nuclei.

博士
國立中山大學
生物醫學研究所
100
Brain stem cardiovascular regulatory dysfunction during brain stem death is underpinned by an upregulation of nitric oxide synthase II (NOS II) in rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from blood pressure of comatose patients that disappears before brain stem death ensues. At the same time, the ubiquitin-proteasome system (UPS) is involved in the synthesis and degradation of NOS II. We assessed the hypothesis that the UPS participates in brain stem cardiovascular regulation during brain stem death by engaging in both synthesis and degradation of NOS II in RVLM. In a clinically relevant experimental model of brain stem death using Sprague-Dawley rats, pretreatment by microinjection into the bilateral RVLM of proteasome inhibitors (lactacystin or proteasome inhibitor II) antagonized the hypotension and reduction in the life-and-death signal elicited by intravenous administration of Escherichia coli lipopolysaccharide (LPS). On the other hand, pretreatment with an inhibitor of ubiquitin-recycling or UCH-L1 potentiated the elicited hypotension and blunted the prevalence of the life-and-death signal. Real-time polymerase chain reaction, Western blot, electrophoresis mobility shift assay, chromatin immunoprecipitation and co-immunoprecipitation experiments further showed that the proteasome inhibitors antagonized the augmented nuclear presence of NF-κB or binding between NF-κB and nos II promoter and blunted the reduced cytosolic presence of phosphorylated IκB. The already impeded NOS II protein expression by proteasome inhibitor II was further reduced after gene-knockdown of NF-κB in RVLM. In animals pretreated with UCH-L1 inhibitor and died before significant increase in nos II mRNA occurred, NOS II protein expression in RVLM was considerably elevated. We conclude that UPS participates in the defunct and maintained brain stem cardiovascular regulation during experimental brain stem death by engaging in both synthesis and degradation of NOS II at RVLM. Our results provide information on new therapeutic initiatives against this fatal eventuality.

博士
國立中山大學
生物科學系研究所
97
As the origin of a “life-and-death” signal that reflects central cardiovascular regulatory failure during brain stem death, the rostral ventrolateral medulla (RVLM) is a suitable neural substrate to evaluate the cellular mechanism of this fateful phenomenon. Based on a clinically relevant animal model that employed the organophosphate pesticide mevinphos (Mev) as the experimental insult, this study evaluated two hypotheses. First, transcriptional upregulation of nitric oxide synthase I or II (NOS I or II) gene expression by nuclear factor-κB (NF-κB) on activation of phosphoinositide 3-kinases (PI3K)/Akt/phosphatase and tensin homologue deleted on chromosome ten (PTEN) cascade in the RVLM underlies brain stem death. Second, muscarinic receptor-independent activation of cyclic adenosine monophosphate-dependent protein kinase A (PKA) in the RVLM is involved in the cardiovascular responses exhibited during Mev intoxication. In Sprague-Dawley rats, our results showed that microinjection bilaterally of Mev (10 nmol) into RVLM induced a progressive augmentation in NF-κB, PI3K, Akt or PTEN activity that paralleled the increase in NOS II or peroxynitrite level in RVLM. Loss-of-function manipulations that included pharmacological blockade, gene knockdown, or immunoneutralization of NF-κB, PI3K or Akt in RVLM significantly potentiated and prolonged the initial increase in “life-and-death” signal, reversed the cardiovascular depression, and blunted the augmented expression of NOS II or nitrotyrosine on induced by Mev. Blockade of PI3K or Akt in RVLM also significantly blunted the Mev-induced activation of NF-κB in the RVLM. However, immunoneutralization of PTEN in RVLM significantly diminished the increase in “life-and-death” signal and potentiated the increase in Akt activity. We conclude that the PI3K/Akt cascade plays a “pro-death” role in our Mev intoxication model of brain stem death by upregulating NF-κB/NOS II/peroxynitrite in the RVLM, subject to antagonism by PTEN in this process. Microinjection bilaterally of Mev (10 nmol) into the RVLM induced a significantly augmentation in PKA activity in ventrolateral medulla that was not antagonized by coadministration of a nonselevtive or selective muscarinic receptor inhibitor. However, pharmacological blockade PKA in RVLM significantly blunted the initial increase in “life-and-death” signal and the accompanying augmentation of NOS I expression in the ventrolateral medulla exhibited during Mev intoxication. We conclude that a muscarinic receptor-independent activation of PKA plays a “pro-life” role in our Mev intoxication model of brain stem death by up regulating NOS I/PKG in the RVLM. According to this study, we proved that Mev stimulates different mechanism, muscarinic receptor-independent/PKA and PI3K/Akt/NF-κB, to regulate NOS I and NOS II expression respectively, and induces cardiovascular responses during “pro-life” and “pro-death” phases. This information should provide further insights on the cellular mechanism of central cardiovascular regulation during the progression towards brain stem death, and offer news vistas in our search for therapeutic remedies or management strategies against fatal organophosphate poisoning and brain stem death.

碩士
國立中山大學
生物科學系研究所
94
Mevinphos (Mev) is an organophosphate insecticide that acts on the rostral ventrolateral medulla (RVLM), the origin of sympathetic vasomotor tone, to induce cardiovascular responses. This study investigated the role of Trk (tropomyosin-related kinase) (receptor form) or Src (non-receptor form) tyrosine kinase at the RVLM in Mev-induced cardiovascular responses. Bilateral microinjection of Mev (10 nmol) into the RVLM elicited two distinct phases of cardiovascular responses, designated Phase I (sympathoexcitatory) and Phase II (sympathoinhibitory) Mev intoxication. Western blot assay showed that whereas p-Trk490 was increased during Phase I, p-Src416 was increased only during Phase II Mev intoxication. Interestingly, application of a Trk specific inhibitor (K252a; 1 pmol) or Src specific inhibitor (SU6656; 100 pmol) into the bilateral RVLM blunted the Mev-elicited sympathoexcitatory or sympathoinhibitory effect, respectively. Besides, K252a was limited to block NOS I protein expression in the RVLM during Mev intoxication, SU6656 only inhibited NOS II protein expression in the RVLM during Mev intoxication. We conclude that Trk tyrosine kinase (p-Trk490) in the RVLM participates in the Phase I cardiovascular responses during Mev intoxication, Src tyrosine kinase (p-Src416) in the RVLM participates in the Phase II cardiovascular responses associated with Mev intoxication.

碩士
國立中山大學
生物科學系研究所
95
We investigated the role of Shc、PYK2、Grb2-sos binding complex、Ras and Raf proteins at the rostral ventrolateral medulla (RVLM), the origin of sympathetic neurogenic vasomotor tone, in mevinphos (Mev) intoxication. Adult Sprague-Dawley rats anesthetized by sodium pentobarbital (45 mg/kg) and maintained by propofol (20-25 mg/kg/hr) were used. Bilateral microinjection of Mev (10 nmol) into the RVLM elicited two distinct phases of cardiovascular responses, designated Phase I (sympathoexcitatory) and Phase II (sympathoinhibitory) Mev intoxication. Pretreatment with microinjection of a phospho-Shc-tyrosine 317, phospho-PYK2-tyrosine 402, phospho-PYK2-tyrosine 579/580 antibody (1:20), Grb2-sos complex inhibitor (SH3b-p), Ras specific inhibitors (manumycin A or FTA) or Raf specific inhibitor (GW5074) into the bilateral RVLM blunted the magnitude of the Mev-elicited sympathoexcitatory cardiovascular effect without affecting the duration. The Mev-elicited sympathoinhibitory cardiovascular effect was not influenced. Our results suggest that signaling pathways that involve Shc, PYK2, Grb2-sos complex, Ras or Raf protein in the RVLM participate in the sympathoexcitatory phase of Mev intoxication.

碩士
慈濟大學
藥理暨毒理學研究所
94
Rostral ventrolateral medulla (RVLM) neurons play an important role in regulation of the cardiovascular function. Microinjection of NMDA into the RVLM produces increases in blood pressure. Our recent study showed that intravenous ethanol selectively inhibited NMDA-induced responses in the RVLM. The inhibitory effects of ethanol were reduced over time during continuous infusion of ethanol, suggesting the development of acute tolerance. Exposure of ethanol has been shown to activate cAMP dependent protein kinase (PKA) signaling pathways and NMDA receptor function is regulated by PKA. We hypothesize that PKA signal may participate in development of acute tolerance to ethanol inhibition of NMDA receptor activation in RVLM. Intravenous injection of ethanol (0.16g) followed by continuous infusion of ethanol (0.16g/hr) into anesthetized SD rats (300~350g) reduced NMDA-induced pressor effect at 10 min, but the inhibition disappeared at 40 min after the injection. The disappearance of ethanol inhibition was dose-dependently blocked by PKA inhibitor KT5720 (40, 400 and 4000 fmol) or a cAMP antagonist cAMPS-Rp (20 and 200 fmol) but not by a cAMP activator cAMPS-Sp (10 pmol), PKC inhibitor chelerythine (10 pmol),and PKC activator phorbol 12-myristate 13-acetate (2 fmol); these agents together with NMDA were microinjection into the RVLM after ethanol. A significant increase in the level of PKA-regulated phosphoserine 897 but not PKC-regulated phosphoserine 896 on NMDA NR1 subunit that was found in rostroventral medulla during acute ethanol tolerance. The results suggest that PKA signaling pathways participate in acute tolerance to ethanol inhibition of NMDA receptor function.

碩士
高雄醫學大學
生理及分子醫學研究所碩士班
94
Sepsis is a common cause of death in the patient of intensive care unit and is the 12th major cause of death in Taiwan. Sepsis is the results of complex systemic inflammatory responses to infection. In recent studies, it was found that hypotension develops in approximately half of the patients with sepsis, and cardiovascular depression is a major cause of inadequate tissue perfusion, leading to multiple organ dysfunctions. Our previous results indicate that nitric oxide (NO) and peroxynitrite (ONOO‾) at rostral ventrolateral medulla (RVLM), where sympathetic promotor neurons for the maintenance of arterial pressure are located, play important roles in the elicitation of cardiovascular depression during endotoxemia. Since NO reacts with superoxide anion (O2‾) to form ONOO‾, and NADPH oxidase is one of the major cellular sources of superoxide, we therefore investigated whether NADPH oxidase-derived superoxide in the RVLM participates in the elicitation of cardiovascular depression after LPS treatment. In adult, male Sprague-Dawley rats (body weight, 270~350 g) that were maintained under propofol anesthesia, microinjection bilaterally into the RVLM of E coli lipopolysaccharide (LPS, 100 ng/50 nl) induced progressive hypotension and bradycardia, alongside significant upregulation of protein expression and enzyme activity of gp91-phox or p47-phox subunit of NADPH oxidase, and increase in superoxide production in the RVLM. Microinjection bilaterally into the RVLM of a superoxide dismutase mimetic, Tempol, (50 nmol) or a NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI, 1.25 nmol), appreciably attenuated the LPS-induced cardiovascular depression and prolonged the survival time. DPI treatment also attenuated the LPS-promoted increase in superoxide production in the RVLM. These results suggest that NADPH oxidase-derived superoxide in the RVLM participates in the elicitation of cardiovascular depression after LPS treatment.

碩士
國立中山大學
生物科學系研究所
96
Peroxisome proliferator activated receptors (PPAR) are members of the nuclear receptor family that act as transcription factors to regulate target gene expression. In addition to their well-known effects in regulation of glucose homeostasis and lipid metabolism, PPAR activators have recently been shown to exert antihypertensive effects, although the underlying mechanism is not clear. Our laboratory has previously demonstrated that oxidative stress of an augmented tissue level of superoxide anion (Ο2•−) in the rostral ventrolateral medulla (RVLM), where promotor neurons for generation of sympathetic vasomotor outflow reside, contributes to neural mechanism of hypertension. I therefore propose to test in my thesis the hypothesis that protection against oxidative stress after activation of the PPARs in the RVLM may contribute to the antihypertensive effect of these transcription factors. Experiments were performed in the spontaneously hypertensive rats (SHR) or normotensive Wistar-Kyoto (WKY) rats under anesthesia or conscious condition. Compared to WKY rats, microinjection bilaterally into the RVLM of a synthetic activator of PPARγ, rosiglitazone (1 nmol), evoked significantly greater decreased in mean systemic arterial pressure (MSAP) and heart rate (HR) in SHR. These cardiovascular suppressive effects of rosiglitazone were accompanied by greater decrease in tissue level of O2 - and upregulation of the antioxidant uncoupling proteins (UCPs) in the RVLM of SHR. Rosiglitazone also caused a significant greater increase in PPARγ expression in the nuclear extracts from RVLM of SHR than WKY rats. All these cellular events induced by rosiglitazone were antagonized by co-administration into the RVLM of the PPARγ inhibitor, GW9662 (5 nmol). This PPARγ inhibitor also significantly reversed the cardiovascular depressive effects of rosiglitazone. Together these results suggest that PPARγ in the RVLM may participate in central cardiovascular regulation by promoting hypotension and bradycardia via amelioration of O2- production and upregulation of antioxidant UCPs. Moreover, a downregulation of the PPARγ in the RVLM may contribute to neural mechanism of hypertension.

碩士
國防醫學院
生理學研究所
82
The periaqueductal grey matter of the midbrain (PAG) is important for defence reactions including increased arterial pressure, vocalization and often attack and filight etc. Medulla oblongata, especially the dorsal medulla (DM) and ventrolateral medulla (VLM), integrates cardiovascular functions. Medulla and PAG, therefore, share a common function regarding cardiovascular integration. The present study is an attempt to study the descending pathway of the pressor mechanism of PAG with reference to that of the DM and VLM and their termination in the intermediolateral column of the upper thoracic spinal cord. 　　In cats anesthetized with a -chloralose (40 mg/kg) and urethane (400 mg/kg) intraperitoneally, the pressor site of PAG, DM or VLM was first identified by rectangular pulses (E., 100-200 uA, 80 Hz, 0.5ms, for 15 sec). The same site in DM or VLM was further confirmed by microinjection of sodium glutamate (Glu, 0.25M, 100 nl) for the induction of vasopressor responses which reflects the presence of perikaryi. The induced increases of systemic arterial pressure (SAP), heart rate (HR) and cardiac contraction (dP/dt) were measured. Since cerebellum may affect the cardiovascular responses of PAG and medulla, in the first set of experiment (n = 6) the effects of cerebellum on the responses of PAG, DM or VLM were determined. It was found that the changes of pressor responses and plasma catecholamines on PAG, DM or VLM activation were not affected after decerebellation through surgical separation of the three cerebellar peduncles. 　　In the second set of experiments (n=23) the changes of cardiovascular responses resulted from PAG, DM and/or VLM activation were compared before, and after electrolytic lesions (DC current, 1 mA for 30 sec) on the ipsilateral and/or contralateral intermediolateral column (IML) of spinal cord at the level of T2. After unilateral IML lesion, the resting cardiovascular-parameters did not show any significant change. The pressor responses of SAP and dP/dt on DM/VLM stimulation by E. and Glu were reduced significantly after ipsilateral IML lesion. The same responses on either E. or Glu activation were not significantly affected by contralateral IML lesion. The PAG-pressor responses were only reduced significantly after bilateral IML lesions. Single lesion on either ipsilateral or contralateral IML was not effective. In the third set of experiment (n=11) the correlation between DM and VLM on both sides of the medulla was determined by longitudinal bisection of the brain stem from upper pons through the upper level of spinal cord. After bisection, the resting cardiovascular parameters and the pressor resposes of DM or VLM were not significantly altered. The fourth set of experiment (n=9) was to determine the correlation of the PAG evoked potential between DM and VLM. It was found that PAG evoked potential at both DM and VLM, but only the late component of the potential correlated well with the induced pressor responses. In this late component the positive and large spike recorded in DM (2.66±0.59 ms) was faster than that of the VLM (40.3±0.66 ms). The fifth set of experiments (n=7) was to determine the changes of extracellular fluid (ECF) catecholamines in DM or VLM during PAG stimulation, occlusions of both carotid arteries (baroreceptor inactivation) and stimulation of the afferent sciatic nerve. It was found that PAG stimulation increased the ECF catecholamines in both DM and VLM. Carotid occlusion and sciatic nerve stimulation increased ECF catecholamines only in DM. The same procedures decreased ECF catecholamines in VLM. 　　Our findings suggest:(1) Cerebellum does not affect the pressor responses induced by activation of the PAG, DM and VLM. (2) Pathways from PAG descend bilaterally, while that of the DM and VLM descend predominantly ipsilaterally to reach the IML at T2. (3) DM is more important for mediating the PAG-pressor responses.

碩士
國立中山大學
生物醫學科學研究所
93
Maintenance of a stable arterial blood pressure is a complex physiological phenomenon. In addition to dysfunction of the blood vessels, alterations in homeostasis of circulating signals and humoral factors also contribute significantly to the development of hypertension. Recent evidence indicates that accumulation of the byproducts of cellular respiration, including superoxide anion (O2-) and/or hydrogen peroxide (H2O2), are contributing factors in pathophysiology of hypertension. With respect to the central nervous system, neurons in the rostral ventrolateral medulla (RVLM) play a pivotal role in neural regulation of blood pressure. RVLM neurons not only provide a tonic excitation to maintain the sympathetic vasomotor activity of the blood vessels, they also participate in baroreceptor reflex control of blood pressure. The notion that production of O2- and/or H2O2 in the RVLM participates in central control of blood pressure has recently gained major recognition in the area of hypertension study. Nonetheless, detailed insights into the mechanisms underlying O2- and/or H2O2 promoted hypertension remain to be elucidated. The hypothesis that forms the basis of this study is that enhanced level of O2- and/or H2O2 in the RVLM may be important factors for the manifestation of hypertension in the spontaneously hypertensive rats (SHR), an animal model of human essential hypertension. In comparison to normotensive Wistar-Kyoto (WKY) rats, basal level of O2- in the RVLM region of adult male SHR rats was significantly higher, along with a reduction in the expression of superoxide dismutase 1 (SOD1), SOD2 or catalase. SOD and catalase are enzymes that metabolize cellular O2- or H2O2 respectively. Pharmacologically, microinjection bilaterally into the RVLM of SOD mimetic, Tempol (50 nmol) or a pan SOD/calatase mimetic, FeTMPyP (100 nmol), significantly decreased mean systemic arterial pressure (MSAP) or heart rate (HR) in both SHR and WKY rats. The maximal hypotensive effect produced by Tempol or FeTMPyP was significantly greater in SHR than WKY rats. We also found that in SHR, but not WKY rats, the hypotensive and bradycardiac responses after microinjection bilaterally into the RVLM of FeTMPyP was significantly greater than that by Tempol. In addition, infection of RVLM neurons with adenoviral vector encoding SOD1 (Ad-SOD1), SOD2 (Ad-SOD2) or catalase (Ad-Catalase) gene (5x108 pfu) into the bilateral RVLM resulted in a long-term hypotensive effect in SHR but not WKY rats. The temporal profile of Ad-catalase-promoted hypotension was again longer than that promoted by Ad-SOD1 or Ad-SOD2 alone. At the molecular level, gene transfer of SOD1, SOD2 or catalase into the RVLM region of SHR or WKY rats specifically increased the expression of individual protein, resulting in a reduction in O2- level. Together these results suggest that accumulation of O2- and/or H2O2 in the RVLM is involved in the neural mechanism of hypertension in SHR.

碩士
國立中山大學
生物科學系研究所
96
Background: The rostral ventrolateral medulla (RVLM), location of the sympathetic premotor neurons, plays a pivotal role in central cardiovascular regulation. The peroxisome proliferator-activated receptors-γ (PPARγ) agonist is commonly prescribed for the treatment of type II diabetes mellitus by its insulin sensitizing ability. Intriguingly, both animal and human studies revealed that PPARγ agonist also possesses blood pressure lowering effect although the underlying mechanism is unknown. We designed a study to evaluate the hypothesis that activation of PPARγ in the RVLM mediates the blood pressure lowering effect of PPARγ agonist, rosiglitazone. Materials and Methods: The 12-week spontaneously hypertensive rats (SHR) and the age-matched normotensive Wistar Kyoto (WKY) rats were used in this study. Basal systemic arterial pressure (SAP) and heart rate (HR) were measured for one week, followed by oral administration of a synthetic PPARγ agonist, rosiglitazone (80 mg/kg/day), or saline for 7 days. The hemodynamic profile was recorded for 4 weeks post treatment. The role of PPARγ in the RVLM on blood pressure lowering effect of rosiglitazone was examined by microinjection bilaterally into the RVLM of the PPARγ antagonist, GW9662 (5 nmol). In a separated series of experiments, the RVLM of SHR or WKY rats was removed at the end of rosiglitazone or saline treatment. Protein expression of PPARα, PPARβ/δ or PPARγ in the RVLM was analyzed by Western blotting. To ascertain that changes in protein expression are not secondary to perturbation of SAP, expression of PPARs was also examined inSHR that received oral administration of a calcium channel inhibitor, amlodipine (16 mg/kg/day), for 7 days. Results: Compared to saline intake, rosiglitazone significantly lowered the mean SBP (MSBP, 159.2±9.9 mmHg vs. 139.8±12.6 mmHg) in SHR, but not WKY rats. This blood pressure lowering effect of rosiglitazone in SHR lasted for at least 10 days post treatment. Rosiglitazone treatment, on the other hand, had no significant effect on HR in SHR or WKY rats. At the end of 7-day treatment, microinjection bilaterally into the RVLM of PPARγ antagonist, GW9662 (5 nmol), significantly reversed the blood pressure lowering effect of rosiglitazone in SHR. In addition, protein expression of PPARα or PPARγ was significantly upregulated in the RVLM of the SHR but not WKY rats that received rosiglitazone treatment. Oral intake of amlodipine (16 mg/kg/day) for 7 days in SHR significantly lowered MSBP (164.8±7.7 mmHg to 131.8±7.8 mmHg), but did not affect protein expression of PPARα, PPARβ/δ or PPARγ in the RVLM of SHR. Conclusion: These results suggest that oral administration of rosiglitazone exerts blood pressure lowering effect via activation of PPARs in the RVLM of SHR. Moreover, upregulation of PPARα or PPARγ in the RVLM may underlie the antihypertensive effect of rosiglitazone.

博士
國立中山大學
生物科學系研究所
93
In eukaryotic cells, most proteins in the cytosol and nucleus are degraded via the ubiquitin-proteasome pathway. Ubiquitin is best known for its role in targeting proteins for degradation by the proteasome. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is found specifically in central and peripheral neurons, and is responsible for the removal of small peptide fragments from the ubiquitin chain and for co-translational processing of ubiquitin gene products to generate free monomeric ubiquitin. In response to extreme conditions, cells exhibit an up-regulation of heat shock protein (HSP) expression, which contributes to repair and protective mechanisms. Within the HSP family, HSP70 is the major inducible member that protects against cell death. Based on the pharmacologic property of organophosphates as an inhibitor of cholinesterase, it is generally contended that manifestations of organophosphate poisoning, including secretion and muscle fasciculation, stupor, cardiopulmonary collapse, respiratory failure, coma or death, result from accumulation of, and over-stimulation by acetylcholine at peripheral of central synapses. One approach in furthering our understanding on organophosphate poisoning is delineation of its potential protective mechanisms. In this regard, the information on the cellular and molecular mechanisms that underlie organophosphate poisoning has received attention. Our laboratory demonstrated previously that a crucial brain site via which mevinphos (Mev), an organophosphate insecticide of the P=O type, acts is the rostral ventrolateral medulla (RVLM), the medullary origin of premotor sympathetic neurons that are responsible for the maintenance of vasomotor tone. The phasic changes in cardiovascular events over the course of acute Mev intoxication also parallel fluctuations of the “life-and-death” signals that emanate form the RVLM. Based on a rat model of organophosphate poisoning that provides continuous information on cellular and molecular mechanisms in the RVLM, the present study was undertaken to evaluate whether changes in protein level of UCH-L1 or HSP70 are associated with death arising from Mev intoxication. We also evaluated the efficacy of both of them in the neuroprotection against fatality during Mev intoxication. The first part of this study investigated whether UCH-L1 plays a neuroprotective role at the RVLM, where Mev acts to elicit cardiovascular toxicity. In Sprague-Dawley rats maintained under propofol anesthesia, Mev (960 µg/kg, i.v.) induced a parallel and progressive augmentation in UCH-L1 or ubiquitin expression at the ventrolateral medulla during the course of Mev intoxication. The increase in UCH-L1 level was significantly blunted on pretreatment with microinjection bilaterally into the RVLM of a transcription inhibitor, actinomycin D (5 nmol) or a translation inhibitor, cycloheximide (20 nmol). Compared to artificial cerebrospinal fluid (aCSF) or sense uch-L1 oligonucleotide (100 pmol) pretreatment, microinjection of an antisense uch-L1 oligonucleotide (100 pmol) bilaterally into the RVLM significantly increased mortality, reduced the duration of the phase I (“pro- life” phase), blunted the increase in ubiquitin expression in ventrolateral medulla, and augmented the induced hypotension in rats that received Mev. The second part of this study investigated whether HSP70 plays a neuroprotective role at the RVLM. Intravenous administration of Mev (960

碩士
國立中山大學
生物科學系研究所
93
The rostral ventrolateral medulla (RVLM) regulates vasomotor activity via sympathoexcitation and sympathoinhibition to maintain blood pressure. Nitric oxide synthesized by nitric oxide synthase (NOS) I and NOS II within RVLM is responsible for sympathoexcitation and sympathoinhibition respectively. In our previously study, under physiological condition RVLM neurons contain both NOS I and NOS II protein, and NOS III protein is expressed mainly on blood vessels. Under Mevinphos (Mev) intoxication, our previously study demonstrates that the expression of RVLM NOS I and II mRNA or protein are both increased under Mev intoxication phase I, and NOSII mRNA or protein are further increased under Mev intoxication phase II. On the other hand, in rat central nervous system, about 65% of total cells are glial cells, including astrocytes, microglia and oligodendrocytes. However, the expressions of NOS isoforms in RVLM glial cells still need to be determined. We used double immunofluorescence staining and confocal microscopy to investigate the distributions of NOS isoforms protein in RVLM neurons and glial cells under physiological condition and under pathological condition using Mev intoxication as our model. We further compared the distributions of NOS isoforms in RVLM neurons and glial cells under physiological or pathological conditions. The confocal images indicate that NOS I protein reactivity co-localized with neurons and microglia in the RVLM. NOS II protein reactivity co-localized with neurons, astrocytes and microglia. NOS III protein reactivity co-localized with blood vessels and microglia. The distributions of NOS isoforms protein reactivity in RVLM neurons and glial cells under Mev intoxication are the same as under physiological condition. Furthermore, the expressions of NOS I protein within neurons or microglia and NOS II in neurons, astrocytes or microglia are progressively increased under Mev intoxication. On the other hand, the expression of NOS III within microglia under Mev intoxication was similar to physiological condition. The population of NOS I-positive neurons or microglia, and NOS II-positive neurons, astrocytes or microglia increased under Mev intoxication. However the population of NOS III-positive microglia decreased under Mev intoxication. These results indicate that within RVLM, the distributions of NOS I are in neurons and microglia; NOS II are in neurons, astrocytes and microglia; NOS III are in blood vessels and microglia. We suggest that under Mev intoxication, the source of up-regulated NOS I protein includes neurons and microglia; and the up-regulated NOS II protein comes from neurons, astrocytes and microglia.

碩士
國立中山大學
生物科學系研究所
97
The rostral ventrolateral medulla (RVLM) is the origin of a “life-and-death” signal identifies from systemic arterial blood pressure spectrum that reflects failure of central cardiovascular regulation during brain stem death. It is also a target site where endogenous angiotensin II acts on angiotensin II type 1 receptors (AT1R) to increase blood pressure (BP); or on type 2 receptors (AT2R) to inhibit baroreceptor reflex (BRR) response. This study investigated the roles of AT1R and AT2R and their signaling pathways in RVLM for “life-and-death” signal response during experimental brain stem death, using organophosphate mevinphos (Mev) as the experimental insult. In Sprague-Dawley rats, Mev (640 μg/kg, i.v.) elicited an increase (pro-life phase) followed by a decrease (pro-death phase). Real-time PCR analysis revealed that whereas AT1R level underwent a 10% increase at pro-life phase, AT2R exhibited a significance increase of up to 40% at pro-death phase. Western blot analysis revealed that whereas AT1R level underwent a 20% increase at pro-life phase, AT2R exhibited a significant increase of up to 50% at pro-death phase. Pretreatment with microinjection of an AT1R antagonist losartan (2 nmol) into RVLM elicited abrupt death because of drastic hypotension through inhibiting NADPH oxidase and its downstream superoxide anion. Pretreatment with NADPH oxidase inhibitor DPI (1.5 nmol) inhibited NADPH oxidase avtiviting and superoxide anion production and decreased “life-and-death” signal at pro-life phase; using superoxide anion inhibitor tempol (5 nmol) potentiated blood pressure and “life-and-death” signal at pro-death phase. However, pretreatment with an AT2R antagonist PD123319 (2 nmol) potentiated the “life-and-death” signal and antagonized hypotension during pro-death phase through inhibiting protein phosphotase 2A (PP2A) then activating extracellular signal-regulated kinase 1/2 (ERK1/2). Similar to AT2R antagonist PD123319, pretreatment with PP2A inhibitor okadaic acid (0.5 fmol) inhibit PP2A, leading to activation of ERK1/2, potentiate “life-and-death” signal and antagonized hypotension during pro-death phase. These results suggest that AT1R in RVLM plays a “pro-life” role through NADPH oxidase/superoxide anion during experimental brain stem death by maintaining BP and “life-and-death” signal; AT2R plays a “pro-death” role through PP2A/ERK1/2 by inhibiting BP and “life-and-death” signal, and superoxide may also plays a “pro-life and pro-death” role at pro-death phase.

博士
國立陽明大學
生理學研究所
87
The present study attempted to decipher the role of glutamatergic neurotransmission at the rostral ventrolateral medulla (RVLM) and the GABAergic neurotransmission at the nucleus tractus solitarii (NTS) in parabrachial nucleus (PBN) complex-induced suppression of cardiac baroreceptor reflex (BRR) response. Experiments were carried out in adult, male Sprague-Dawley rats that were anesthetized and maintained with pentobarbital sodium (15-20 mg/kg/h, i.v.). The sensitivity of BRR control of heart rate (HR) was evaluated by measuring the reflex bradycardia in response to a transient hypertension evoked by an i.v. bolus administration of phenylephrine (5 mg/kg). The quotient calculated from the peak reflex decrease in HR for a given peak increase in mean systemic arterial pressure (MSAP) (beat/min/mmHg) was used as the index for cardiac BRR response. Distribution of loci in the PBN complex from which activation induced suppression of reflex bradycardia and of the projecting neurons to the RVLM and the NTS Under a stimulus condition that did not appreciably alter the baseline MSAP and HR, electrical (10-s train of 0.5-ms pulses, at 10-20 mA ad 10-20 Hz) or chemical (L-glutamate, 1 nmol) stimulation of the ventrolateral regions and Kolliker-Fuse (KF) subnucleus of PBN complex significantly suppressed the reflex bradycardia (> 30%). To compare the distribution of PBN neurons projecting to the NTS and the RVLM, two major integrating areas of the BRR loop, rats were subject to microinjection of Fast Blue and Diamidino Yellow into the ipsilateral NTS and RVLM, respectively. Both kinds of retrogradely labeled neurons were distributed primarily in the KF subnucleus and the surrounding ventrolateral areas of the PBN complex. They were intermixed with each other with very few double-labeled neurons in these regions of the PBN complex. These results suggest that the PBN complex may participate in central cardiovascular regulation by inhibiting the BRR response. In addition, it is likely that two distinct descending pathways to the NTS and RVLM may be involved in the suppressive action of the PBN complex. The role of glutamatergic neurotransmission at the rostral ventrolateral medulla in parabrachial nucleus-induced baroreflex suppression The aim of this study was to investigate whether glutamatergic neurotransmission in the RVLM is involved in the suppression of cardiac BRR response by PBN complex. On the basis of in vivo microdialysis coupled with high-performance liquid chromatography-fluorescence detection of amino acid, we found that repeated activation of PBN complex (5-min train of 0.5-ms rectangular pulses, at 10-20 mA and 10-20 Hz) increased the extracellular concentration of glutamate in the dialysate collected from the RVLM. The same stimulus condition also produced a temporal related suppression of the cardiac BRR response. Microinfusion of L-glutamate (50 mM) via the microdialysis probe into the RVLM caused an increase in the extracellular glutamate concentration. The same treatment also significantly inhibited the cardiac BRR response. On the other hand, the PBN-induced suppression of reflex bradycardia was appreciably reversed by bilateral microinjection into the RVLM of the N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (500 pmol), or the non-NMDA receptor antagonist, CNQX (50 pmol). Anatomically, most of the retrogradely labeled neurons in the ventrolateral regions and KF subnucleus of the ipsilateral PBN complex after microinjection of Fast Blue into the RVLM were also immunoreactive to anti-glutamate antiserum. These results suggest that glutamate released from the PBN glutamatergic projection to the RVLM may participate in PBN-induced suppression of cardiac BRR response by activating both NMDA and non-NMDA receptors at the RVLM. The role of GABAergic neurotransmission at the nucleus tractus solitarii in phasic and tonic suppression of BRR response by parabrachial nucleus complex We then investigated the role of GABAergic neurotransmission at the NTS in PBN complex-induced suppression of cardiac BRR response. Utilizing the same experimental procedures, we found that repeated activation of PBN complex (15 min trains of 0.5-ms rectangular pulses, at 10-20 mA and 10-20 Hz) resulted in a site-specific increase in GABA concentration in the dialysate collected from the NTS. The temporal course of this increase in the NTS coincided with the time course of inhibition on the BRR response elicited by the PBN complex. Bilateral microinjection of lidocaine (5%, 300 nl) to reversibly block synaptic transmission at the ventrolateral regions and KF subnucleus of PBN complex elicited an enhancement of the cardiac BRR response. The temporal course of this facilitatory effect correlated positively with the time course of decrease in extracellular GABA concentration in the NTS. Moreover, we found that inhibition of cardiac BRR response by the PBN complex was reversed by bilateral microinjection into the NTS of the GABAA receptor antagonist, bicuculline methiodide (5 pmol), or the GABAB receptor antagonist, 2-OH saclofen (500 pmol). Anatomically, many of the retrogradely labeled neurons in the KF subnucleus and surrounding areas of the PBN complex after microinjection of Fast Blue into the NTS were immunoreactive to anti-glutamic acid decarboxylase (GAD) antiserum. These results suggest that GABA released from the NTS may participate in PBN-induced suppression of cardiac BRR response phasically and tonically by activating both the GABAA and GABAB receptors at the NTS. Furthermore, a direct GABAergic descending projection from the KF subnucleus and surrounding areas of the PBN complex to the NTS may be involved in this tonic cardiovascular modulation. GABAergic neurotransmission at the nucleus tractus solitarii participates in glutamate-mediated baroreflex suppression at the rostral ventrolateral medulla In this series of experiments we investigated the role of GABAergic neurotransmission at the NTS in glutamate-mediated suppression of cardiac BRR response when administered into the RVLM. We found that microinfusion of L-glutamate (50 mM) via the microdialysis probe into the RVLM caused an increase in the extracellular glutamate concentration. The same treatment also resulted in an increase in GABA concentration in the dialysate collected from the NTS. The temporal course of this increase in extracellular GABA concentration in the NTS coincided with the time course of inhibition elicited by microinfusion of L-glutamate on the cardiac BRR response. On the other hand, suppression of cardiac BRR response to L-glutamate (100 pmol), microinjected into the RVLM, was also appreciably reversed by bilateral NTS microinjection of the GABAA receptor antagonist, bicuculline methiodide (5 pmol), or the GABAB receptor antagonist, 2-OH saclofen (500 pmol). These results suggest that GABA released from the NTS may participate in glutamate-mediated suppression of cardiac BRR response when administered into the RVLM. In addition, both the GABAA and GABAB receptors at the NTS may participate in this process. Conclusion On the basis of in vivo microdialysis, pharmacological manipulation in conjunction with neuroanatomical evaluation, the present study demonstrated that the parabrachial descending projections to the RVLM and the NTS from the KF subnucleus and the surrounding ventrolateral regions may participate in the suppression of cardiac BRR by this pontine nucleus. Under a normal condition, GABA released from a direct PBN-NTS GABAergic nerve terminal may function as a chemical mediator to tonically suppress the cardiac BRR response. Superimposed upon this operating scheme, activated PBN complex may enhance its suppressive action on BRR response via recruitment of an indirect neural pathway to the RVLM by increasing glutamate release in this nucleus to activate both NMDA and non-NMDA receptors. Glutamate released in the RVLM may also activate the RVLM-NTS connection to cause an increase in GABA release in the NTS. The augmented GABA concentration at the NTS may participate in the phasic suppression of cardiac BRR by PBN complex via activation of both GABAA and GABAB receptors at the NTS. These results demonstrate the physiologic significance of PBN complex in central cardiovascular regulation. More importantly, they also improve our understanding of neural circuitry of the glutamatergic neurotransmission at the RVLM and GABAergic neurotransmission at the NTS as well as their interplay in the suppression of baroreceptor reflex response by the PBN complex.

碩士
國立中山大學
生物科學系研究所
98
Insulin resistacne and hyperinsulinemia are important risk factors for development of type 2 diabetes mellitus and hypertension. Recently, accumulating evidence has shown that endothelial dysfunction, increases in peripheral vessel resistnce and overactivation of the sympathetic neruvous system contribute to the development of insulin resistance-associated hypertension. The signigicance of cardiovascular regulatory center in the brain stem in pathophysiology of the insulin resistance-induced hypertension, however, has not been explored. Previously studies have proved that increases in superoxide anion (O2˙−) production in peripheral tissue and suppression of nitric oxide (NO) expression in the endothial cell are involved in insulin resistance and hypertension. The nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (RVLM) are involved in neural regulation of blood pressure by serving respectively as the primary baroreceptor afferent terminal sites and the location of sympathetic premotor neurons for cardiovascular regulation in the brain stem. Clinically, the peroxisome proliferator-activated receptor (PPAR) agonist is commonly prescribed for the treatment of type 2 diabetes mellitus by activate PPARγ to enhance peripheral tissue insulin sensitizing ability, to maintain blood glucose homeostasis. Intriguingly, both animal and human studies revealed that PPARγ agonist also possesses blood pressure lowering effect, although the underlying mechanism is not clear. We therefore investigated in the present study the role of NO and O2˙− in the NTS and RVLM in the pathophysiology of the high fructose diet-induced insulin resistacne and hypertension, and to evaluate the potential central antihypertensive effect of PPARγ agonist in rats subjected to high fructose diet. The normotensive male Wistar Kyoto rats (WKY) were divided into 4 groups, including 3 experimental group that received 60% high fructose diet for 8 weeks and one control group that received regular chow diet for the same period of time. Within the 3 experimental groups, two of them received oral administration of rosiglitazone or pioglitazone (10 mg/kg/day) at the last two weeks (from week 6 to week 8) and the third group received saline ingestion. Systemic blood pressure was measured by tail vein sphygmomanometer very week and venous blood was drawn every other week to measure blood sugar and insulin level. At the end of the experiment, oral glucose tolerance test (OGTT) was tested and O2˙− and NO production in the NTS and RVLM were quantified. In adult male WKY rats I found that high fructose diet induced insulin resistance, hypertriglycemia and hypertension. Oral administration of rosiglitazone or pioglitazone significantly blunted the hypertension, hypertriglyceridemia, and ameliorated insulin resistance induced by high fructose diet. The high fructose diet also increased tissue level of O2˙− in the NTS and RVLM. PPARγ agonist treatment for two weeks did not affect the induced oxidative stress in these two nuclei. NO production was also increased in the NTS and RVLM after high fructose diet for 6 weeks. Oral treatment of rosiglitazone or pioglitazone significantly attenuated NO production after high fructose diet. At the molecular level, protein expressions of the NADPH oxdase subunits (p40phox, p47phox and gp91phox) and superoxide dismutase (cupper/zinc SOD, mitochondrial SOD, extracellular SOD) were not altered in the NTS or RVLM after high fructose diet alone or in addition with rosiglitazone or pioglitazone treatment. In the RVLM, there was a significant increase in neuronal NO synthase (nNOS) expression with concomitant decrease in inducible NOS (iNOS) expression. Oral treatment of PPARγ agonist for two weeks significantly suppressed the induced nNOS upregulation and attenuated the induced downregulation of iNOS expression in the RVLM. Together these results suggest that overproduction of O2˙− and NO in the NTS and RVLM may related to the development of insulin resistance-associated hypertension. Oral treatment of PPARγ agonist, including rosiglitazone and pioglitazone, may provide antihypertensive protection by superssing the induced-nNOS expression and increasing the induced-iNOS expression in the RVLM.

碩士
中原大學
醫學工程學系
87
The present project studies the effects of chemical lesioning in dorsal and ventrolateral medulla on the glutamate-induced cardiovascular action of gigantocellular tegmental field and lateral field in cats. Beside observation of the responses about systemic arterial pressure(SAP), heart rate and indirect cardiac contractility induced by microinjection of sodium glutamate in these regions, spectral analysis of the vertebral nerve activity(VNA) with fast fourier transform(FFT) was used to characterize the effects of these regions on VNA. Experiments were performed on 35 adult cats of both sexes weighing between 2.5 and 4.0 kg. These cats were anesthetized by intraperitoneal injection a mixture of -chloralose(40mg/kg) and urethane(400mg/kg), and artificially ventilated to maintain the end-tidal CO2 at approximately 4%. The femoral artery was cannulated for monitoring the systemic arterial blood pressure(SAP) and heart rate(HR). Peripheral sympathetic nerve activity was recorded from the central end of the left postganglionic verterbral nerve. Potential were recorded monophasically under mineral oil with a bipolar platinum electrode. The nerve signal was amplified with the band pass filter set at 1Hz and 1000Hz. Microinjections were made from a glass micropipette held in a stereotaxic carrier. The volume ranged from 50 to 70 nl for monosodium glutamate(Glu, 0.25mM dissolved in artifical cerebrospinal fluid with 0.5% pontamine sky blue). Glu was microinjection into the pontine gigantocellular tegmental field(PFTG) or pontine lateral tegmental field(PLTF). The dorsal medulla(DM) or rostral ventrolateral medulla(RVLM) was lesioned by microinjection of 150-200 nl of kainic acid ( 24mM dissolved with 0.5% pontamine sky blue in artifical cerebrospinal fluid) Finding of the present experiments showed that when the ipsilateral DM was lesioned, microinjection of Glu into PFTG no longer elicited pressor response. On the contrary, when the contralateral DM was lesioned, the Glu-induced of PFTG responses were little affected. Lesion of the ipsil- or contralateral RVLM slightly affected the Glu-induced of PFTG responses. This suggests that connection between PFTG and DM in producing pressor action is principally unilaterally. Glu stimulation of PLTF produced bradycardia associated with slight hypotension. When the ipsilateral or contralateral DM was lesioned, microinjection of Glu into PLTF no longer elicited depressor response. On the other hand, when the ipsilateral or contralateral RVLM was lesioned, the same microinjection of the Glu induced responses in PLTF was converted from depressor to pressor response. This suggests that PLTF may also contain sympathoexcitatory neurons. In conclusion, our data indicate that the PFTG contains sympathoexcitatory neurons, but the area of PLTF contains a mixture of sympathoexcitatory and parasympathoexcitatory neurons