Cerebral Cortical Vasodilation via Nicotinic Receptors by Heated Tobacco Product Aerosol Extract in Rats.

INTRODUCTION: Smoking increases the risk of lung cancer due to a number of components of smoke. The use of novel heated tobacco products (HTPs), alternative to conventional combustion cigarettes, has increased in recent years. However, the in vivo biological effects of HTPs are poorly understood. This study aimed to clarify the acute effects of injecting aerosol extract prepared from an HTP on regional cerebral blood flow (rCBF) in rat cortex by comparing them to the effects of injecting smoke extract prepared from conventional combustible cigarettes. METHODS: In urethane anesthetized rats, rCBF was measured using laser speckle contrast imaging simultaneously with arterial pressure. RESULTS: Both cigarette smoke extract and HTP aerosol extract, at a dose equivalent to 30 µg nicotine/kg, injected intravenously, increased cortical rCBF without changing arterial pressure. The magnitude and time course of the increased rCBF response to both extracts were similar throughout the cortical area, and the rCBF increases were all abolished by dihydro-ß-erythroidine, an α4ß2-preferring nicotinic acetylcholine receptor (nAChR) antagonist. CONCLUSION: In conclusion, our study demonstrated that the effect of injecting aerosol extract prepared from an HTP, an acute increase in cortical rCBF, is mediated via activation of α4ß2-like neuronal nAChRs in the brain.

Influence of age on nicotinic cholinergic regulation of blood flow in rat's olfactory bulb and neocortex.

The olfactory bulb receives cholinergic basal forebrain inputs as does the neocortex. With a focus on nicotinic acetylcholine receptors (nAChRs), this review article provides an overview and discussion of the following findings: (1) the nAChRs-mediated regulation of regional blood flow in the neocortex and olfactory bulb, (2) the nAChR subtypes that mediate their responses, and (3) their activity in old rats. The activation of the α4ß2-like subtype of nAChRs produces vasodilation in the neocortex, and potentiates olfactory bulb vasodilation induced by olfactory stimulation. The nAChR activity producing neocortical vasodilation was similarly maintained in 2-year-old rats as in adult rats, but was clearly reduced in 3-year-old rats. In contrast, nAChR activity in the olfactory bulb was reduced already in 2-year-old rats. Thus, age-related impairment of α4ß2-like nAChR function may occur earlier in the olfactory bulb than in the neocortex. Given the findings, the vasodilation induced by α4ß2-like nAChR activation may be beneficial for neuroprotection in the neocortex and the olfactory bulb.

Nicotinic cholinergic regulation of olfactory bulb blood flow response in aged rats.

In our previous research, we had demonstrated the crucial role of neuronal nicotinic acetylcholine receptors (nAChRs) in potentiation of the olfactory bulb blood flow response to olfactory stimulation in adult rats. The present study examined the effects of nAChR activation on the olfactory bulb blood flow response in rats aged 24-27 months. We found that, under urethane anesthesia, unilateral olfactory nerve stimulation (300 µA, 20 Hz, 5 s) increased blood flow within the ipsilateral olfactory bulb, without changes in the systemic arterial pressure. The increase in blood flow was dependent upon the current and frequency of the stimulus. Intravenous administration of nicotine (30 µg/kg) had little effect on the olfactory bulb blood flow response to nerve stimulation at either 2 Hz or 20 Hz. These results suggest a reduction in nAChR-mediated potentiation of the olfactory bulb blood flow response in aged rats.

Olfactory function and discrimination ability in the elderly: a pilot study.

We recently reported that subjects with a higher olfactory identification threshold for rose odor declined more in attentional ability in the elderly. This study focuses on discrimination ability and olfactory identification threshold in twelve elderly subjects living in a community (age: 80.9 ± 1.6). Olfactory function was assessed by the rose odor identification threshold. We assessed the discrimination ability by distinguishing 5 similar odor pairs. Our results showed that the subjects with a higher olfactory identification threshold (≥ 5) declined more in discrimination ability (14% ± 14%, p = 0.03) compared to those with a lower threshold (≤ 4) (averaged value set at 100%). As discrimination ability is related to the basal forebrain cholinergic system, our results suggest that olfactory impairment links to the decline in cognitive function relating the cholinergic system.

The relationship between olfaction and cognitive function in the elderly.

This study investigated the relationship between olfaction and cognitive function in 12 elderly people (age: 80.9 ± 1.6) living in the community. Olfactory function was assessed by the identification threshold for rose odor. Four cognitive measures consisting general cognitive ability assessed by Mini-Mental State Examination (MMSE), its sub-domains, and attentional ability assessed by drawing a line to connect the numbers consecutively (trail-making test part A; TMT-A), were assessed. Subjects with a higher olfactory threshold (≥ 5) declined more in the performance speed of TMT-A (73% ± 7%, p = 0.05) compared with those subjects with a lower threshold (≤ 4) (averaged value was set at 100%). Other cognitive statuses assessed by MMSE tended to decline in subjects with higher thresholds. Because attentional function relates to the basal forebrain cholinergic system, our results suggest that olfactory impairment links to the decline in cognitive function, particularly of attention-relating cholinergic function.

Effects of nicotine on regional blood flow in the olfactory bulb in response to olfactory nerve stimulation.

This study examined the effect of olfactory nerve stimulation on regional cerebral blood flow and assessed the effect of intravenous nicotine administration on this response in anesthetized rats. Regional cerebral blood flow was measured with laser Doppler flowmetry or laser speckle contrast imaging. Unilateral olfactory nerve stimulation for 5 s produced current (≥ 100 µA) and frequency-dependent (≥ 5 Hz) increases in blood flow in the olfactory bulb ipsilateral to the stimulus. The increased olfactory bulb blood flow peaked at 30 ± 7% using stimulus parameters of 300 µA and 20 Hz. Nerve stimulation did not change frontal cortical blood flow or mean arterial pressure. The intravenous injection of nicotine (30 µg/kg) augmented the olfactory bulb blood flow response to nerve stimulation (20 Hz, 300 µA) by approximately 1.5-fold (60-s area after the stimulation). These results indicate that olfactory nerve stimulation increases olfactory bulb blood flow, and the response is potentiated by the activation of nicotinic cholinergic transmission.

Somatosensory regulation of resting muscle blood flow and physical therapy.

Somatosensory stimulation can affect skeletal muscle blood flow (MBF) at rest in anesthetized animals via pressor reflex response or antidromic and local vasodilation. Increase in MBF due to reflex pressor response occurs generally in the skeletal muscles of the entire body, while antidromic and local vasodilation are limited to the peripheral stimulation site. Since increased MBF improves several disorders (muscle stiffness, pain, etc.), it is reasonable to further explore the effective use of somatic stimulation in physical therapies, such as massage, acupuncture, anma, and shiatsu or acupressure, in treating skeletal muscle disorders.

Neural Mechanisms Involved in the Noxious Physical Stress-Induced Inhibition of Ovarian Estradiol Secretion.

Stress is known to change the secretion of ovarian steroid hormones via the hypothalamic-pituitary-ovarian (HPO) axis. Noxious physical stress can cause reflex responses in visceral function via autonomic nerves. This article reviews our recent animal studies on neural mechanisms involved in ovarian estradiol secretion induced by noxious physical stress stimulation. In anesthetized rats, noxious physical stress (pinching the hindpaw or electrical stimulation of the tibial nerve) decreased ovarian estradiol secretion. These noxious stress-induced ovarian hormonal responses were observed after decerebration but were abolished after spinal transection. Electrical stimulation of the ovarian sympathetic nerves (superior ovarian nerves: SON) decreased ovarian estradiol secretion. The reduced secretion of ovarian estradiol induced by hindpaw pinching was abolished by bilateral severance of the SON. Efferent activity of the SON was increased following hindpaw pinching. Thus, the inhibition of ovarian estradiol secretion during noxious physical stress was mainly integrated in the brainstem, and this inhibitory response was due to reflex activation of sympathetic nerves to the ovary. In rats, the sympathetic inhibitory regulation of ovarian estradiol secretion was pronounced when the HPO axis was inhibited by chronic estradiol treatment. Considering the female life cycle, extensive physical stress may inhibit ovarian function, especially before puberty and during old ages when the HPO axis is inactive. Anat Rec, 302:904-911, 2019. © 2019 Wiley Periodicals, Inc.

Effects of nicotine on odor-induced increases in regional blood flow in the olfactory bulb in rats.

The present study examined the effects of nicotinic acetylcholine receptor activation on the odor-induced blood flow response in the olfactory bulb. In urethane-anesthetized rats, odor stimulation (5% amyl acetate, 30 s) produced an increase in olfactory bulb blood flow (reaching 107% ± 3% of the pre-stimulus basal values), without changes in frontal cortical blood flow or mean arterial pressure. Intravenous injection of nicotine (30 µg/kg), a nicotinic acetylcholine receptor agonist, significantly augmented the odor-induced increase response of olfactory bulb blood flow, without changes in the basal blood flow level. The nicotine-induced augmentation of the olfactory bulb blood flow response to odor was negated by dihydro-ß-erythroidine, an α4ß2-preferring nicotinic acetylcholine receptor antagonist. Our results suggest that the activation of α4ß2-like neuronal nicotinic acetylcholine receptors in the brain potentiates an odor-induced blood flow response in the olfactory bulb.

Blood pressure-independent increase in the cortical cerebral blood flow induced by manual acupuncture of the auricular region in rats.

We aimed to determine whether acupuncture to the auricular region increases cortical regional cerebral blood flow (rCBF). The rCBF was measured using laser speckle contrast imaging in urethane-anesthetized rats. Acupuncture stimulation was performed manually at the auricular concha or abdomen. The former's stimulation significantly increased the rCBF of the bilateral cerebral cortex in the frontal, parietal, and occipital lobes without altering the systemic arterial pressure. In contrast, abdominal stimulation affected neither rCBF nor systemic arterial pressure. The increase in the rCBF was completely abolished by the severance of the somatic nerves that innervated the auricular region, comprising the trigeminal nerve, facial nerve, auricular branch of the vagal nerve, glossopharyngeal nerve, and great auricular nerve. Thus, application of acupuncture to the auricular region increases the rCBF without increasing arterial pressure.

Effect of basal forebrain stimulation on extracellular acetylcholine release and blood flow in the olfactory bulb.

The olfactory bulb receives cholinergic basal forebrain input, as does the neocortex; however, the in vivo physiological functions regarding the release of extracellular acetylcholine and regulation of regional blood flow in the olfactory bulb are unclear. We used in vivo microdialysis to measure the extracellular acetylcholine levels in the olfactory bulb of urethane-anesthetized rats. Focal chemical stimulation by microinjection of L-glutamate into the horizontal limb of the diagonal band of Broca (HDB) in the basal forebrain, which is the main source of cholinergic input to the olfactory bulb, increased extracellular acetylcholine release in the ipsilateral olfactory bulb. When the regional cerebral blood flow was measured using laser speckle contrast imaging, the focal chemical stimulation of the HDB did not significantly alter the blood flow in the olfactory bulb, while increases were observed in the neocortex. Our results suggest a functional difference between the olfactory bulb and neocortex regarding cerebral blood flow regulation through the release of acetylcholine by cholinergic basal forebrain input.

Mechanism of physical stress-induced inhibition of ovarian estradiol secretion in anesthetized rats.

This study examined the site of main integration center in the physical stress-induced inhibition of ovarian estradiol secretion because of ovarian sympathetic nerve (superior ovarian nerve: SON) activation in anesthetized rats. In central nervous system-intact rats, electrical stimulation of the tibial afferent nerve at 10V increased the efferent activity of the SON by 39±13% and reduced the ovarian secretion of estradiol by 34±7%. These responses were observed in decerebrate rats but were abolished in spinal rats. Thus, the main integration center for this ovarian hormonal response is located in the brain stem.

Somatoautonomic reflexes in acupuncture therapy: A review.

Oriental therapies such as acupuncture, moxibustion, or Anma, have been used to treat visceral disorders since ancient times. In each of these therapies, stimulation of the skin or underlying muscles leads to excitation of afferent nerves. The sensory information is carried to the central nervous system, where it is transferred to autonomic efferents, thus affecting visceral functions. This neuronal pathway, known as the "somatoautonomic reflex", has been systematically studied by Sato and his colleagues for over a half century. Nearly all their studies were conducted in anesthetized animals, whereas human patients are conscious. Responses in patients or the events following therapeutic somatic stimulation may differ from those observed in anesthetized animals. In fact, it is increasingly apparent that the responses in patients and animals are not always coincident, and the differences have been difficult for clinicians to reconcile. We review the mechanism of the "somatoautonomic reflex" as described in anesthetized animals and then discuss how it can be applied clinically.

Systemic blood pressure alters cortical blood flow and neurovascular coupling during nociceptive processing in the primary somatosensory cortex of the rat.

Inference on nociceptive and pain-related processes from functional magnetic resonance imaging is made with the assumption that the coupling of neuronal activity and cerebral hemodynamic changes is stable. However, since nociceptive stimulation is associated with increases in systemic arterial pressure, it is essential to determine whether this coupling remains the same during different levels of nociception and pain. The main objective of the present study was to compare the amplitude of local field potentials (LFP) and cerebral blood flow (CBF) changes in the primary somatosensory cortex during nociceptive electrical stimulation of the contralateral or ipsilateral forepaw in isoflurane-anesthetized rats, while manipulating mean arterial pressure (MAP). MAP changes induced by nociceptive stimulation were manipulated by transecting the spinal cord at the upper thoracic segments (T1-T2), which interrupts sympathetic pathways and prevents nociception-related MAP increases, while sensory pathways between the forepaws and the brain remain intact. Intensity-dependent increases in MAP and CBF were observed and these effects were abolished or significantly decreased after spinal transection (p<0.001 and p<0.05, respectively). In contrast, the intensity-dependent changes in LFP amplitude were decreased for the contralateral stimulation but increased for the ipsilateral stimulation after spinal transection (p<0.05). Thus, neurovascular coupling was altered differently by stimulus-induced MAP changes, depending on stimulus intensity and location. This demonstrates that CBF changes evoked by nociceptive processing do not always match neuronal activity, which may lead to inaccurate estimation of neuronal activity from hemodynamic changes. These results have important implications for neuroimaging of nociceptive and pain-related processes.

Stimulus frequency-dependent inhibition of micturition contractions of the urinary bladder by electrical stimulation of afferent Aß, Aδ, and C fibers in cutaneous branches of the pudendal nerve.

We aimed to examine the afferent mechanisms for the reflex inhibition of the rhythmic micturition contractions (RMCs) of the urinary bladder induced by stimulation of the perineal skin afferents in urethane-anesthetized rats. Electrical stimulation (pulse duration: 0.5 ms) was applied to the cutaneous branches of the pudendal nerve (CBPN) at frequencies of 0.1, 1, and 10 Hz for 1 min. Nerve fiber groups were defined by recording compound action potentials from CBPN. Activation of only Aß fibers (0.2 V) produced an inhibition of RMCs at 7-11 min after the onset of stimulation (late inhibition), at any tested frequency. Additional activation of Aδ fibers (1 V) produced additional early inhibition (immediately after stimulation) at 1 and 10 Hz. Furthermore, additional activation of C fibers (10 V) at 10 Hz completely stopped RMCs for >10 min. This strong inhibition persisted after local application of capsaicin to the stimulating CBPN. We conclude that activities of Aß, Aδ, and C afferent fibers, without capsaicin-sensitive channels, can contribute to the inhibition of bladder contractions.

Selective Expression of Osteopontin in ALS-resistant Motor Neurons is a Critical Determinant of Late Phase Neurodegeneration Mediated by Matrix Metalloproteinase-9.

Differential vulnerability among motor neuron (MN) subtypes is a fundamental feature of amyotrophic lateral sclerosis (ALS): fast-fatigable (FF) MNs are more vulnerable than fast fatigue-resistant (FR) or slow (S) MNs. The reason for this selective vulnerability remains enigmatic. We report here that the extracellular matrix (ECM) protein osteopontin (OPN) is selectively expressed by FR and S MNs and ALS-resistant motor pools, whereas matrix metalloproteinase-9 (MMP-9) is selectively expressed by FF MNs. OPN is secreted and accumulated as extracellular granules in ECM in three ALS mouse models and a human ALS patient. In SOD1(G93A) mice, OPN/MMP-9 double positivity marks remodeled FR and S MNs destined to compensate for lost FF MNs before ultimately dying. Genetic ablation of OPN in SOD1(G93A) mice delayed disease onset but then accelerated disease progression. OPN induced MMP-9 up-regulation via αvß3 integrin in ChAT-expressing Neuro2a cells, and also induced CD44-mediated astrocyte migration and microglial phagocytosis in a non-cell-autonomous manner. Our results demonstrate that OPN expressed by FR/S MNs is involved in the second-wave neurodegeneration by up-regulating MMP-9 through αvß3 integrin in the mouse model of ALS. The differences in OPN/MMP-9 expression profiles in MN subsets partially explain the selective MN vulnerability in ALS.

Sympathetic regulation of ovarian functions under chronic estradiol treatment in rats.

Activation of the sympathetic nerve to the ovary (superior ovarian nerve: SON) decreases ovarian blood flow and estradiol secretion in rats in the estrous phase. The present study examined the effects of long-term estradiol treatment on the sympathetic regulation of both ovarian blood flow and estradiol secretion. Non-pregnant Wistar rats received sustained subcutaneous estradiol (5µg/day) or saline for 4weeks. Chronic estradiol treatment did not affect ovarian blood flow at rest, while changed the basal ovarian estradiol secretion rate, i.e., narrow ranges (4-34pg/min) in estradiol-treated rats, versus wide ranges (3-192pg/min) in saline-treated rats of different estrous cycles. SON was electrically stimulated at different frequencies (2, 5 and 20Hz). Ovarian blood flow was decreased by SON stimulation in a stimulus frequency-dependent manner in both saline- and estradiol-treated rats, but the threshold was shifted from 2Hz to 5Hz after chronic estradiol treatment. Ovarian estradiol secretion rate was not significantly changed by SON stimulation at any frequency in saline-treated rats, while it was markedly decreased by SON stimulation at high frequencies (5 and 20Hz) in estradiol-treated rats. In conclusion, chronic estradiol treatment augments sympathetic inhibition of ovarian estradiol secretion perhaps by inhibiting the hypothalamic-pituitary-ovarian axis.

Afferent fibers involved in the bradykinin-induced cardiovascular reflexes from the ovary in rats.

Bleeding or rupture of the ovary often accompanies ovarian cysts and causes severe pain and autonomic responses such as hypotension. It would be expected that ovarian afferents contribute to cardiovascular responses induced by ovarian failure. The present study examined cardiovascular responses to noxious chemical stimulation of the ovary by bradykinin, an algesic substance released by tissue damage, and explored the role of ovarian afferents in the ovarian-cardiovascular responses in anesthetized rats. Non-pregnant adult rats were anesthetized with pentobarbital and artificially ventilated. The carotid artery was cannulated to monitor blood pressure and heart rate. Noxious chemical stimulation was achieved by applying a small piece of cotton soaked with bradykinin to the surface of the ovary for 30s. Application of bradykinin (10(-4) M) to the ovary decreased heart rate and blood pressure. These cardiovascular responses were not significantly influenced by severance of the vagal nerves or the superior ovarian nerve, but were abolished by severance of the ovarian nerve plexus (ONP). Application of bradykinin (10(-4) M) to the ovary evoked afferent activity of the ONP both in vivo and in vitro preparations. These results indicate that the decreases in heart rate and blood pressure following chemical noxious stimulation of the ovary with bradykinin are reflex responses, whose afferent nerve pathway is mainly through afferent fibers in the ONP.