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Hypertension is the leading risk factor for cardiovascular morbidity and mortality, and growing evidence implicates chronic renal inflammation as a culprit in the development of hypertension, such as in the case of the autoimmune disease systemic lupus erythematosus (SLE). Our lab uses the established female NZBWF1 mouse model of SLE to mechanistically link renal inflammation to development of hypertension. Mice are typically shipped to our university young (6 weeks), develop autoantibodies as adults (~20 weeks), and are hypertensive by 35 weeks. We observed that mice shipped while young in summer months have higher renal cortical heat shock protein (HSP)90 immediately upon arrival compared to those shipped in winter. Summer mice also have higher autoantibodies and albuminuria in adulthood than winter mice. These findings suggest that seasonal and environmental factors like heat, compounded by travel stress, act as early life stressors that adversely affect disease progression in these mice. However, it is unknown how seasonal travel stressors affect HSP90, SLE disease progression, and blood pressure in mice exposed as adults rather than in early life. We aim to study if seasonal factors affect adult-traveling mice differently and if HSP90 is associated with these differences. We hypothesize that summer travel will upregulate HSP90 in adult female lupus mice, leading to increases in disease severity and blood pressure compared to winter mice. Female NZBWF1 mice were shipped in either summer or winter at 18–19 weeks. At 35 weeks, mice were implanted with carotid catheters to assess mean arterial pressure (MAP) for two days. Mice were euthanized, with blood and kidneys collected to assess plasma double-stranded (ds)DNA autoantibodies as well as plasma and renal levels of HSP90. Adult-shipped mice developed heightened dsDNA autoantibodies (5.4e5±5.8e4 vs. 3.8e5±5.5e4 U/mL; P=0.0423; n=13–19), plasma HSP90 (36.42±6.41 vs. 21.59±2.29 pg/mL; P=0.0082; n=15–20), and renal cortical HSP90 (0.16±0.02 vs. 0.03±0.01 pg/μg; P NIH (K01HL139869, R01HL153703), Departmental Seed Grant, and Department of Defense (LR210096) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Systemic lupus erythematosus (SLE) is an autoimmune disorder linked to chronic renal inflammation and prevalent hypertension. We use an established murine model of SLE (NZBWF1) to mechanistically link renal inflammation to the development of hypertension. SLE mice travel to our university pre-puberty (3-6 weeks), develop autoantibodies by 20 weeks, and are consistently hypertensive by 35 weeks. We observed that when mice travel in the summer, autoantibody production is higher as the mice age vs. those that travel in the winter. This association was heightened in a subset of SLE mice that traveled to our university as adults (18-19 weeks), suggesting a role of seasonal factors, travel stressors, and age in altering disease severity. It is unknown whether these seasonal and environmental stressors can also alter blood pressure and inflammatory processes. Thus, we hypothesized that travel-induced seasonal stressors exacerbate hypertension and renal inflammation in adult SLE-prone mice. Female SLE mice traveled to our university in summer or winter at 3-6 or 18-19 weeks and were implanted with carotid catheters once they reached 35 weeks to assess mean arterial pressure (MAP). Mice were then euthanized and tissues collected to monitor plasma and renal interleukin (IL)-6, a proinflammatory cytokine. No seasonal differences were found in MAP of mice that traveled pre-puberty in summer (n = 17) and winter (n = 39) (143.6 ± 3.5 vs. 140.2 ± 2.2 mmHg; p = 0.4096) nor in plasma IL-6 (26.06 ± 7.25 vs. 15.54 ± 8.622 pg/mL; p = 0.2370) or renal IL-6 (0.01156 ± 0.00603 vs. 0.01099 ± 0.00523 pg/μg; p = 0.9445). Conversely, adult SLE mice that traveled in summer had higher MAP than those that traveled in winter (136.12 ± 2.63 vs. 128.81 ± 1.55 mmHg; p = 0.0339; n = 5-7), as well as higher plasma IL-6 (723.28 ± 220.72 vs. 280.73 ± 67.99 pg/mL; p = 0.0339) and renal IL-6 (0.078956 ± 0.017192 vs. 0.023413 ± 0.003801 pg/μg; p < 0.0001). Together, our data indicate that hypertension and renal inflammation in mice subjected to travel stress as adults is seasonally dependent. Since there were no seasonal differences in hypertension in SLE mice that traveled pre-puberty, there may be a factor related to age, seasonal environmental factors, and/or timing of the stressors that is causing the detrimental effect.

Renal inflammation promotes hypertension in systemic lupus erythematosus (SLE). Our recent studies suggest that renal inflammation is regulated by the vagally-mediated cholinergic anti-inflammatory pathway, and that vagal tone is reduced in SLE mice. We found that augmenting central bioavailability of acetylcholine via the acetylcholinesterase inhibitor, galantamine, increases vagal tone and reduces renal inflammation and mean arterial pressure (MAP) in SLE mice after two weeks. However, the long-term efficacy and therapeutic potential of galantamine are unknown. Thus, we hypothesized that chronic galantamine administration would effectively halt the progression of SLE hypertension and renal injury. Starting at 20 weeks of age, female SLE ( NZBWF1 ) and control ( NZW ) mice received galantamine hydrobromide (3 mg/kg/day, SQ) or vehicle continuously for 16 weeks via osmotic minipump. At 35 weeks, mice were placed in metabolic cages for urine collection, carotid catheters were implanted to assess MAP in conscious mice, and tissues were harvested. MAP (mmHg, n=4-6) was higher in SLE mice than controls (161.9±9.9 vs. 128.3±0.9, p=0.001) and long-term galantamine treatment prevented further increases in MAP in SLE mice (161.9±9.9 vs. 138.3±4.9, p= 0.01). Survival rate was lower in SLE mice compared to controls (50% vs. 100%) and galantamine improved survival to 70% in SLE mice. Eighty percent (4 of 5) of SLE mice had urinary albumin over 300 mg/dL and chronic galantamine lowered the prevalence to 57% (4 of 7); no controls had albuminuria. There was no difference in CD3+CD4+ T cells amongst groups, but the percentage of renal CD3+CD8+ T cells was increased in SLE mice compared to controls (29.7±2.2 vs. 12.8±0.79, p

Systemic lupus erythematosus (SLE) is characterized by hypertension that results from chronic renal inflammation and dysautonomia in the form of dampened vagal tone. In health, the vagus nerve regulates inflammatory processes through mechanisms like the cholinergic anti-inflammatory pathway; so in the case of SLE, reduced efferent vagus nerve activity may indirectly affect renal inflammation and therefore hypertension. In this study, we sought to investigate the impact of disrupting vagal neurotransmission on renal inflammation and hypertension in the setting of chronic inflammatory disease. Female SLE ( NZBWF1) and control ( NZW) mice were subjected to a right unilateral cervical vagotomy or sham surgery and 3 wk later were implanted with indwelling catheters to measure blood pressure. Indices of splenic and renal inflammation, as well as renal injury, were assessed. Unilateral vagotomy blunted SLE-induced increases in mean arterial pressure, albumin excretion rate, and glomerulosclerosis. This protection was associated with reduced splenic T cells and attenuated SLE-induced increases in renal proinflammatory mediators. In summary, these data indicate that unilateral vagotomy reduces renal inflammation and reduces blood pressure in SLE mice. The vagus nerves have myriad functions, and perhaps other neuroimmune interactions compensate for the ligation of one nerve.

The cholinergic anti-inflammatory pathway is a vagally-mediated mechanism that controls inflammation. Our published data suggest that an impaired cholinergic anti-inflammatory pathway contributes to hypertension and renal disease in female mice with systemic lupus erythematosus (SLE), since pharmacological potentiation of the efferent vagus via administration of galantamine reduces renal inflammation, mean arterial pressure (MAP) and glomerulosclerosis. The aim of the current study is to selectively target neurons within the dorsal motor nucleus of the vagus (DMV) to stimulate the efferent vagus and the cholinergic anti-inflammatory pathway using designer receptors exclusively activated by designer drugs (DREADDs). We hypothesized that selective activation of DMV neurons would reduce inflammation, eliminating the associated end organ damage in SLE. To study this, female SLE ( NZBWF1 ) and parental control ( NZW ) mice received bilateral microinjections of pAAV-hSyn-hM3D(Gq)-mCherry or pAAV-hSyn-mCherry (control virus) into the DMV at 31 weeks of age using the following coordinates with calamus as reference: 0 mm caudal, 0.25 mm lateral and 0.48 mm ventral. Two weeks post-microinjection, DREADD agonist CNO (3mg/kg) was administered subcutaneously for 2 weeks starting at 33 weeks. At 35 weeks, mice were housed in metabolic cages for urine collection and catheters were implanted in the carotid artery for MAP measurement. Mice were subsequently euthanized and the brain collected to confirm the site of virus microinjection. Selective activation of DMV neurons decreased the incidence of albuminuria [> 300 mg/dL; 66% (4 out of 6) vs. 0% (0 out of 7)], urinary leukocytes [62.5% (5 out of 8) vs. 50% (3 out of 6)] and blood in the urine [50% (4 out of 8) vs. 16% (1 out of 6)] in SLE mice. MAP did not significantly change with the chemogenetic activation of DMV neurons in SLE mice or parental controls (SLE/control virus: 146 ± 6, n=7; SLE/Gq DREADD: 142 ± 3, n=6; NZW/control virus: 126 ± 4, n=4; NZW/Gq DREADD: 132 ± 2, n=5). These results suggests that this timeline of selective activation of DMV neurons in SLE mice reduces renal injury without altering blood pressure, but future studies will confirm the effect on hypertension and renal inflammation in SLE mice

Arginine vasopressin (AVP) and oxytocin (OXY) are released by magnocellular neurosecretory cells that project to the posterior pituitary. While AVP and OXY currently receive more attention for their contributions to affiliative behavior, this mini-review discusses their roles in cardiovascular function broadly defined to include indirect effects that influence cardiovascular function. The traditional view is that neither AVP nor OXY contributes to basal cardiovascular function, although some recent studies suggest that this position might be re-evaluated. More evidence indicates that adaptations and neuroplasticity of AVP and OXY neurons contribute to cardiovascular pathophysiology.

There is a critical need for safe treatment options to control inflammation in patients with systemic lupus erythematosus (SLE) since the inflammation contributes to morbidity and mortality in advanced disease. Endogenous neuroimmune mechanisms like the cholinergic anti-inflammatory pathway can be targeted to modulate inflammation, but the ability to manipulate such pathways and reduce inflammation and end organ damage has not been fully explored in SLE. Positive allosteric modulators (PAM) are pharmacological agents that inhibit desensitization of the nicotinic acetylcholine receptor (α7-nAChR), the main anti-inflammatory feature within the cholinergic anti-inflammatory pathway, and may augment α7-dependent cholinergic tone to generate therapeutic benefits in SLE. In the current study, we hypothesize that activating the cholinergic anti-inflammatory pathway at the level of the α7-nAChR with systemic administration of a partial agonist, GTS-21, and a PAM, PNU-120596, would reduce inflammation, eliminating the associated end organ damage in a mouse model of SLE with advanced disease. Further, we hypothesize that systemic α7 ligands will have central effects and improve behavioral deficits in SLE mice. Female control (NZW) and SLE mice (NZBWF1) were administered GTS-21 or PNU-120596 subcutaneously via minipumps for 2 weeks. We found that the increased plasma dsDNA autoantibodies, splenic and renal inflammation, renal injury and hypertension usually observed in SLE mice with advanced disease at 35 weeks of age were not altered by GTS-21 or PNU-120596. The anxiety-like behavior presented in SLE mice was also not improved by GTS-21 or PNU-120596. Although no significant beneficial effects of α7 ligands were observed in SLE mice at this advanced stage, we predict that targeting this receptor earlier in the pathogenesis of the disease may prove to be efficacious and should be addressed in future studies.

RATIONALE: Effector memory T lymphocytes (T(EM) cells) exacerbate hypertension in response to repeated hypertensive stimuli. These cells reside in the bone marrow for prolonged periods and can be reactivated upon re-exposure to the hypertensive stimulus. OBJECTIVE: Because hypertension is associated with increased sympathetic outflow to the bone marrow, we hypothesized that sympathetic nerves regulate accumulation and reactivation of bone marrow residing hypertension-specific T(EM) cells. METHODS AND RESULTS: Using unilateral superior cervical ganglionectomy in wild-type C57BL/6 mice, we showed that sympathetic nerves create a bone marrow environment that supports residence of hypertension-specific CD8(+) T cells. These cells, defined by their proliferative response upon co-culture with dendritic cells from angiotensin II infused mice, were reduced in denervated compared to innervated bone of angiotensin II-infused mice. Adoptively transferred CD8(+) T cells from angiotensin II-infused mice preferentially homed to innervated compared to denervated bone. In contrast, ovalbumin responsive T cells from OT-I mice did not exhibit this preferential homing. Increasing superior cervical ganglion activity by activating Gq-coupled DREADD (designer receptor exclusively activated by designer drug) augmented CD8(+) T(EM) bone marrow accumulation. Adoptive transfer studies using mice lacking β2 adrenergic receptors (β2AR) indicate that β2AR in the bone marrow niche, rather than T cell β2AR is critical for T(EM) cell homing. Inhibition of global sympathetic outflow using Gi-coupled DREADD injected into the rostral ventrolateral medulla or treatment with a β2AR antagonist reduced hypertension specific CD8(+) T(EM) cells in the bone marrow and reduced the hypertensive response to a subsequent response to low dose angiotensin II. CONCLUSIONS: Sympathetic nerves contribute to the homing and survival of hypertension-specific T(EM) cells in the bone marrow after they are formed in hypertension. Inhibition of sympathetic nerve activity and β2AR blockade reduces these cells and prevents the blood pressure elevation and renal inflammation upon re-exposure to hypertension stimuli.

Diabetes promotes renal sympathetic hyperactivity, autonomic imbalance, and cardiovascular and renal dysfunction. Bilateral renal denervation (BRD) has emerged as a treatment for diabetes; however, the mechanisms that underlie the beneficial effects of BRD are unknown. Aims The present study evaluated the effects of BRD on autonomic, cardiovascular, metabolic, and renal function in streptozotocin-diabetic rats. Main methods Wistar rats were separated into three experimental groups: control (CTR), diabetic (DM), and diabetic that underwent BRD (DM BRD). BRD was performed two weeks after STZ-diabetes induction, the experiments were performed four weeks after DM induction. This study evaluated sympathetic vasomotor nerve activity in different territories (renal, lumbar and splanchnic), arterial baroreceptor reflex, metabolic and renal function. Key findings BRD significantly reduced glycemia, glycosuria, albuminuria, and SGLT2 gene expression in the kidney in DM rats. Renal sympathetic nerve activity (rSNA) was significantly increased and splanchnic sympathetic nerve activity (sSNA) was significantly decreased in DM rats, without changes in lumbar sympathetic nerve activity (lSNA). BRD was able to normalize sSNA and significantly increase lSNA in DM rats compared to control rats. Additionally, cardiac baroreceptor sensitivity was impaired in DM rats, and BRD significantly improved baroreflex sensitivity. Significance Our data suggest that renal nerves play an important role in autonomic, cardiovascular, and renal dysfunction in STZ-DM rats. Thus, sympathetic renal hyperactivity should be considered a possible therapeutic target in diabetic patients.

Background: Mesenchymal stem cells (MSC) improve renal function and renovascular hypertension in the 2-kidney 1-clip model (2K-1C). While MSC play an immunomodulatory role, induce neoangiogenesis, and reduce fibrosis, they do not correct sodium loss by the contra­lateral kidney. Objectives: We investigated the tubular function of both stenotic and contralateral kidneys and the effect of MSC treatment by evaluating diuresis, natriuresis, and the expression of the main water and sodium transporters. Method: Adult Wistar rats were allocated into four groups: control (CT), CT+MSC, 2K-1C, and 2K-1C+MSC. MSC (2 × 105) were infused through the tail vein 3 and 5 weeks after clipping. Systolic blood pressure (SBP) was monitored weekly by plethysmography. Six weeks after clipping, 24-hour urine and blood samples were collected for biochemical analysis. Gene expression of the Na/H exchanger-3, epithelial sodium channel, Na/K-ATPase, Na/K/2Cl cotransporter, and aquaporins 1 and 2 (AQP1 and AQP2) were analyzed by RT-PCR. Intrarenal distribution of AQP1 and AQP2 was analyzed by immunohistochemistry. Results: In hypertensive 2K-1C animals, MSC prevented additional increases in BP. AQP1, but not AQP2, was suppressed in the contralateral kidney, resulting in significant increase in urinary flow rate and sodium excretion. Gene expressions of sodium transporters were similar in both kidneys, suggesting that the high perfusing pressure in the contralateral kidney was responsible for increased natriuresis. Contralateral hypertensive kidney showed signs of renal deterioration with lower GFR in spite of normal RPF levels. Conclusions: MSC treatment improved renal function and enhanced the ability of the contralateral kidney to excrete sodium through a tubular independent mechanism contributing to reduce SBP.

Mesenchymal stem cells (MSC) induced neovascularization and improved renal morphology of the stenotic kidney in 2 kidneys-1 clip (2K-1C) model of renovascular hypertension. The present study evaluated the effects of MSC in the contralateral hypertensive kidney. Three weeks after left renal artery occlusion, MSC were injected into the tail vein of the 2K-1C rats. Renal function and morphology were analyzed in both kidneys. Labeled MSC were found in stenotic and contralateral kidneys. Hypertensive 2K-1C animals presented increased circulating levels of Angiotensin II (Ang II) and renin. MSC prevented the progressive increase of blood pressure and reduced circulating Ang II and renin levels. Stenotic kidney showed reduced renal plasma flow (RPF) and glomerular filtration rate (GFR), whereas the contralateral kidney had a tendency (p > 0.5) of reduction in GFR in spite of unchanged RPF. MSC treatment caused an improvement in GFR with no effect of on RPF in the stenotic kidney. Contralateral kidney showed increased diuresis and natriuresis that were even higher in MSC-treated animals, indicating that cell treatment improved the capacity of the contralateral kidney to excrete sodium. Contralateral kidney expressed higher levels of inflammatory cytokines (IL-6, TNF-α) and signs of fibrosis, which were attenuated by MSC treatment. MSC treatment improved the stenotic kidney function, and it was also beneficial to the contralateral hypertensive kidney because it improved the morphology and preserved its capacity to excrete sodium.

BACKGROUND Although angiotensin II (Ang II) is essential to the development of renovascular hypertension, aldosterone plays a role as well. Recent studies have demonstrated a cross-talk between Ang II type 1 and mineralocorticoid receptors in the brain and kidneys. However, the role of aldosterone in the autonomic and renal dysfunction of renovascular hypertension is not well understood. AIM The current study evaluated whether aldosterone contributes to cardiovascular and renal dysfunction in the 2 kidney-1 clip (2K1C) model. METHODS Mean arterial pressure (MAP) and baroreceptor reflex for control of the heart rate were evaluated in 2K1C treated or not treated with spironolactone (200mg/kg/day, 7 days). Tonic and reflex control of renal sympathetic nerve activity (rSNA) were assessed in urethane-anaesthetized rats. Plasma renin activity (PRA), kidney renin protein expression, renal injury, and central AT1 receptor protein expression were assessed. RESULTS Spiro reduced MAP (198±4 vs. 170±9mm Hg; P < 0.05), normalized rSNA (147±9 vs. 96±10 pps; P < 0.05), and increased renal baroreceptor reflex sensitivity in the 2K1C rats. Spiro reduced α-smooth muscle actin expression in the nonclipped kidney in the 2K1C group (5±0.6 vs. 1.1±0.2%; P < 0.05). There was no change in PRA; however, a decrease in renin protein expression in the nonclipped kidney was found in the 2K1C treated group (217±30 vs. 160±19%; P < 0.05). Spiro treatment decreased AT1 receptor in the central nervous system (CNS) only in 2K1C rats (138±10 vs. 84±12%; P < 0.05). CONCLUSION Aldosterone contributes to autonomic dysfunction and intrarenal injury in 2K1C, these effects are mediated by the CNS.

Sympathetic vasomotor activity is significantly increased in renovascular hypertension. Renal denervation (DnX) has emerged as a novel therapy for resistant hypertension to drug therapy. However, the underlying mechanisms regarding the reduction in blood pressure (BP) after DnX remain unclear. Thus, the aim of this study was to evaluate the effects of DnX of a clipped kidney on the baseline and baroreceptor reflex control of post-ganglionic sympathetic activity to the contralateral kidney (rSNA) and lumbar (lSNA) nerves in Goldblatt hypertensive rats (2K1C). Renal denervation of an ischaemic kidney (DxX - all visible bundles of nerves were dissected - 10% phenol) was performed 5weeks after clipping (gap width: 0.2mm). Ten days after DnX, BP was significantly reduced (16%) in the 2K1C compared with the undenervated 2K1C (p

BACKGROUND Renovascular hypertension (2-kidney 1-clip model (2K1C)) is characterized by renin-angiotensin system (RAS) activation. Increased Angiotensin II (AngII) leads to sympathoexcitation, oxidative stress, and alterations in sodium and water balance. AIM The aim of this study was to evaluate whether a discrete increase in sodium chloride intake in 2K1C rats leads to changes in cardiovascular and autonomic function, oxidative stress, and renin angiotensin aldosterone system. METHODS After 4 weeks of induction of hypertension, rats were fed a normal sodium diet (0.4% NaCl) or a high-sodium diet (2% NaCl) for 2 consecutive weeks. Experiments were carried out for 6 weeks after clipping. Mean arterial pressure (MAP), renal sympathetic nerve activity (rSNA), arterial baroreflex control of rSNA, and heart rate (HR) were assessed. Thiobarbituric acid reactive substances and glutathione were measured as indicators of systemic oxidative stress. Angiostensin-converting enzyme (ACE), ACE2, and angiotensinogen were evaluated in clipped and unclipped kidneys as also urinary angiotensinogen and plasma renin activity. Angiotensinogen, plasma renin activity (PRA) and angiotensin-converting enzyme (ACE) and ACE2 in clipped and unclipped kidneys were evaluated. RESULTS High-sodium diet did not change systemic oxidative stress, and basal values of MAP, HR, or rSNA; however, increased renal (-0.7±0.2 vs. -1.5±0.1 spikes/s/mm Hg) and cardiac (-0.9±0.14 vs. -1.5±0.14 bpm/mm Hg) baroreceptor reflex sensitivity in 2K1C rats. Although there was no alteration in PRA, a high-salt diet significantly decreased urinary angiotensinogen, ACE, and ACE2 expressions in the clipped and unclipped kidneys. CONCLUSIONS Increased arterial baroreceptor control associated with a suppression of the intrarenal RAS in the 2K1C rats on high-salt diet provide a salt-resistant effect on hypertension and sympathoexcitation in renovascular hypertensive rats.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:30:51Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:38:05Z : No. of bitstreams: 1 2-s2.0-84884377371.pdf: 853558 bytes, checksum: fe3ed7b99cb6ec15c0f43fb9e74b985f (MD5) Made available in DSpace on 2014-05-27T11:30:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-10-11 The knowledge of the mechanisms underlying circulating volume control may be achieved by stretching a balloon placed at the junction of the superior vena cava-right atrial junction (SVC-RAJ). We investigated whether the inflation of a balloon at the SVC-RAJ inhibits the intake of 0.3M NaCl induced by GABAA receptor activation in the lateral parabrachial nucleus (LPBN) in euhydrated and satiated rats. Male Wistar rats (280-300g) with bilateral stainless steel LPBN cannulae and balloons implanted at the SVC-RAJ were used. Bilateral injections of the GABAA receptor agonist muscimol (0.5ηmol/0.2l) in the LPBN with deflated balloons increased intake of 0.3M NaCl (30.1±3.9 vs. saline: 2.2±0.7)ml/210min, n=8) and water (17.7±1.9 vs. saline: 2.9±0.5ml/210min). Conversely, 0.3M NaCl (27.8±2.1ml/210min) and water (22.8±2.3ml/210min) intake were not affected in rats with inflated balloons at the SVC-RAJ. The results show that sodium and water intake induced by muscimol injected into the LPBN was not affected by balloon inflation at the SVC-RAJ. We suggest that the blockade of LPBN neuronal activity with muscimol injections impairs inhibitory mechanisms activated by signals from cardiopulmonary volume receptors determined by balloon inflation. © 2013 The Authors. Department of Physiology Institute of Bioscience São Paulo State University (UNESP), 18618-000 Botucatu, SP Department of Physiology and Pathology School of Dentistry São Paulo State University (UNESP), 14801-903 Araraquara, SP Department of Physiology Institute of Bioscience São Paulo State University (UNESP), 18618-000 Botucatu, SP Department of Physiology and Pathology School of Dentistry São Paulo State University (UNESP), 14801-903 Araraquara, SP